Isotopically labeled bile acid derivatives

ABSTRACT

The present application relates to isotopically labeled compounds of Formula I and methods of preparation and use thereof

BACKGROUND

Selective incorporation of isotopes (e.g., deuterium in place ofhydrogen) has a unique effect of retaining the biochemical potency andselectivity of physiologically active compounds while modifyingmetabolic properties to alter their overall therapeutic profile. In somecases, this modification has the potential to have a positive impact onsafety, efficacy and tolerability. Isotopically labeled (e.g.,deuterated and/or radiolabeled) compounds have been widely studied inclinical and non-clinical settings and used in humans as metabolic orpharmacokinetic probes.

Bile acids (BAs) are well known for their role in the solubilization anddigestion of lipid-soluble nutrients. Recently, BAs have emerged assignaling molecules with systemic endocrine functions. BAs andderivatives thereof have been shown to modulate several nuclear hormonereceptors, notably the farnesoid X receptor (FXR), and are agonists forthe G protein-coupled receptor TGR5. Signaling via FXR and TGR5modulates several metabolic pathways, regulating not only BA synthesisand enterohepatic recirculation, but also triglyceride, cholesterol,glucose and energy homeostasis (Thomas, et al. Nat Rev Drug Discovery,2008, 7, 678-693).

A semi-synthetic bile acid analogue, 3α,7α-dihydroxy-6α-ethyl-5β-cholan-24-oic acid (6-ethyl-chenodeoxycholicacid (6-ECDCA) or obeticholic acid (OCA)), disclosed in WO 2002/75298 isa highly potent FXR modulator, which is currently marketed as OCALIVA®for the treatment of primary biliary cholangitis (PBC).

Another semi-synthetic bile acid analogue, 3α,7α,11β-trihydroxy-6α-ethyl-5β-cholan-24-oic acid (compound 100) while beinga potent FXR agonist, also showed specificity against G protein-coupledreceptor TGR5 (GP-BAR1, M-BAR, GPBAR, or GPR131).

Accordingly, new isotopically labeled bile acid derivatives are neededto further investigate the important medical benefits of this class ofcompounds and enhance their clinical safety, tolerability and/orefficacy.

SUMMARY

The present disclosure relates to isotopically labeled (e.g., deuteratedand/or radiolabeled) derivatives of obeticholic acid including aminoacid conjugates and glucuronides thereof.

In some of the embodiments the present disclosure pertains to a compoundof Formula I.

or a pharmaceutically acceptable salt thereof, wherein R₁, R₂, R₃, R₄,R₅, R₆, R₁₁, R₁₆, R₂₃, Z_(1a), Z_(1b), Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₅, Z₆, Z₇, Z₈, Z₉, Z_(11a), Z_(11b), Z₁₂, Z₁₄, Z_(15a),Z_(15b), Z_(16a), Z_(16b), Z₁₇, Z₁₈, Z₁₉, Z₂₀, Z₂₁, Z_(22a), Z_(22b),Z_(23a), Z_(23b), Z_(24a), Z_(24b), and Z₂₅ are as described herein.

In some embodiments, the present disclosure relates to a compound ofFormula II

Some embodiments of the present disclosure relate to a compound ofFormula III

In some embodiments, the present disclosure relates to a compound ofFormula IV

In some embodiments, the present disclosure relates to a compound ofFormula V

In some embodiments, the present disclosure relates to compounds ofFormula VI

In some embodiments, the present disclosure relates to compounds ofFormula VII

wherein

-   -   R₁, R₂, R₃, R₄, R₁₁, R₁₆, and R₂₃ are as described above. In        some embodiments, the present disclosure relates to a process        for preparing the compounds of Formula I.

Some embodiments of the present disclosure pertain to a pharmaceuticalcomposition comprising the compound of Formula I and a pharmaceuticallyacceptable diluent, excipient or carrier.

Some embodiments of the present disclosure pertain to a method ofmodulating FXR activity in a subject in need thereof, comprisingadministering a therapeutically effective amount of the compound ofFormula I.

Some embodiments of the present disclosure pertain to a method ofmodulating TGR5 activity in a subject in need thereof, comprisingadministering a therapeutically effective amount of the compound ofFormula I.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent application, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art. In the case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be limiting.

Other features and advantages of the application will be apparent fromthe following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the ¹H NMR spectrum obtained from d₅-OCA.

FIG. 2 shows the ¹H NMR spectrum obtained from d₇-OCA.

FIG. 3 shows the ¹H NMR spectrum obtained from d₂-OCA (C23(d₂)-OCA).

FIG. 4 shows the ¹H NMR spectrum obtained from d₄-OCA(C3(d)-C7(d)-C23(d₂)-OCA).

FIG. 5 shows the ¹H NMR spectrum obtained from deuterated glycineconjugate of d₅-OCA, d₅-OCA-O (d₂)Gly.

FIG. 6 shows the ¹H NMR spectrum obtained from deuterated taurineconjugate of d₅-OCA, d₅-OCA-O(d₄)Tau.

FIG. 7 shows the ¹H NMR spectrum obtained from OCA-3-O-Glucuronide.

FIG. 8 shows the ¹H NMR spectrum obtained from [³H]-OCA-24-Glucuronide.

FIG. 9 shows comparison plot for ¹H NMR spectra of OCA-24-Glucuronidemethyl ester and [³H]OCA-24-Glucuronide (expansion of 3.0-5.0 ppmregion).

FIG. 10 shows the ¹H NMR spectrum obtained from [¹⁴C]OCA.

FIG. 11 shows the ¹H NMR spectrum obtained from OCA Reference Material.

FIG. 12 is a graph showing Mean (SD) Plasma Concentrations of TotalRadioactivity from 0 to 72 Hours Following IV 100 μg Microtracer Dose of[¹⁴C] OCA, Log and Linear Scales, Part 1-PK Population (N=5).

FIG. 13 is a graph showing Mean (SD) Plasma Concentrations of OCA from 0to 72 and 0 to 24 Hours Following IV 100 μg Microtracer Dose of[¹⁴C]-OCA, Log Scale, Part 1-PK Population (N=5).

FIG. 14 is a graph showing Mean (SD) Plasma Concentrations of OCA from 0to 24 and 0 to 72 Hours Following 25 mg Oral Dose of OCA, Log Scale,Part 1-PK Population (N=5).

FIG. 15 is a graph showing Mean (SD) Plasma Concentrations of Total OCAfrom 0 to 72 Hours Following 25 mg Oral Dose of OCA, Linear and Logscales, Part 1—PK Population (N=5).

FIG. 16 is a graph showing Mean (SD) Plasma Concentrations of Total OCAand Total Radioactivity Following a 25 mg Oral Dose of [¹⁴C]-OCA from 0to 72 Hours, Linear and Log Scales, Part 2, PK Population (N=8).

FIG. 17 is a graph showing Mean (SD) Plasma Concentrations of Total OCAand Total Radioactivity Following a 25 mg Oral Dose of [¹⁴C]-OCA, 4-WeekSampling Period, Part 2, Linear and Log Scales, PK Population (N=8).

FIG. 18 is a graph showing Mean (SD) Cumulative Amount Excreted, MassBalance of Urine, Fecal and Total Excretion and Recovery, Study Part 2,Mass Balance Population, (N=8).

DETAILED DESCRIPTION Definitions

As used in this disclosure and the accompanying claims, the indefinitearticles “a” and “an” and the definite article “the” include plural aswell as single referents, unless the context clearly indicatesotherwise.

As used herein, the phrase “a compound of the invention” refers to acompound of any one of Formula I, II, III, IV, V, VI, VII or VIII or anycompound explicitly disclosed herein.

The present disclosure relates to isotopically-labeled compounds ofFormula I, wherein one or more atoms are replaced by an atom having anatomic mass or mass number different from the atomic mass or mass numberusually found in nature. Examples of isotopes that can be incorporatedinto compounds of the disclosure include isotopes of hydrogen, carbon,nitrogen, and oxygen. Unless otherwise stated, when a position isdesignated specifically as “H” or “hydrogen”, the position is understoodto have hydrogen at its natural abundance isotopic composition or itsisotopes, such as deuterium (d, d, D, or ²H) or tritium (³H).

In some embodiments, isotopically labeled compounds of the presentdisclosure (e.g., those labeled with ²H, ³H and/or ¹⁴C or ¹³C) areuseful in compound and/or substrate tissue distribution assays.Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C) isotopes are useful fortheir ease of preparation and detectability. Further, substitution withheavier isotopes such as deuterium (i.e., ²H or d) may afford certaintherapeutic advantages resulting from greater metabolic stability (e.g.,increased in vivo half-life or reduced dosage requirements) and hencemay be preferred in some circumstances. In some embodiments, inisotopically labeled compounds of the present disclosure any carbon canbe ¹¹C, ¹²C, ¹³C, or ¹⁴C, any nitrogen atom can be ¹³N, ¹⁴N or ¹⁵N, andany oxygen atom can be ¹⁵O, ¹⁶O, ¹⁷O¹⁷O or ¹⁸O.

Deuterium is a safe, nonradioactive relative of hydrogen that can beisolated from sea water and has been used extensively in human metabolicand clinical studies. The average adult human body contains about 1-2grams of deuterium due to its general abundance in nature.

The term “deuterated” as used herein alone or as part of a group, meanssubstituted deuterium atoms. The term “deuterated analog” or “deuteratedcompound” as used herein alone or as part of a group, means substituteddeuterium atoms in place of hydrogen. The deuterated analog of thedisclosure may be a fully or partially deuterium substituted derivative.In some embodiments, the deuterium substituted derivative of thedisclosure holds a fully or partially deuterated substituent, e.g.,alkyl group.

A deuterated drug is a medicinal product (e.g., compound of Formula I)in which one or more of the hydrogen atoms contained in the drugmolecule have been replaced by deuterium. A compound of the invention ora pharmaceutically acceptable salt or solvate thereof that contains theaforementioned deuterium atom(s) is within the scope of the invention.Further, substitution with heavier deuterium, i.e., ²H, can affordcertain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements. Isotopically labeled compounds of the present disclosurecan generally be prepared by following procedures analogous to thosedescribed herein, including Schemes and Examples disclosed herein, bysubstituting an isotopically labeled reagent for a non-isotopicallylabeled reagent.

Deuterium-containing drugs, because of the kinetic isotope effect, mayhave significantly lower rates of metabolism, and hence a longerhalf-life. The present invention also comprehends deuterium labeledcompounds of Formula I where one or more hydrogen atoms is replaced by adeuterium atom having an abundance of deuterium at that position that issubstantially greater than the natural abundance of deuterium, which is0.015%.

The term “deuterium enrichment factor” as used herein means the ratiobetween the deuterium abundance and the natural abundance of adeuterium. In one aspect, a compound of the invention has a deuteriumenrichment factor for each deuterium atom of at least 3500 (52.5%deuterium incorporation at each deuterium atom), at least 4000 (60%deuterium incorporation), at least 4500 (67.5% deuterium incorporation),at least 5000 (75% deuterium), at least 5500 (82.5% deuteriumincorporation), at least 6000 (90% deuterium incorporation), at least6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuteriumincorporation), at least 6600 (99% deuterium incorporation), or at least6633.3 (99.5% deuterium incorporation).

“Specific activity” as used herein means the activity per quantity of aradionuclide and is a physical property of that radionuclide. Activityis a quantity related to radioactivity. The SI unit of activity is thebecquerel (Bq), equal to one reciprocal second. The becquerel is howmany radioactive transformations per second occur in a radioactiveisotope. Its related and more common unit is the Curie (abbreviated Ci)which is 3.7×1010 transformations per second. Since the probability ofradioactive decay for a given radionuclide is a fixed physical quantity(with some slight exceptions, see Changing decay rates), the number ofdecays that occur in a given time of a specific number of atoms of thatradionuclide is also a fixed physical quantity (if there are largeenough numbers of atoms to ignore statistical fluctuations). Thus,specific activity is defined as the activity per quantity of atoms of aparticular radionuclide. It is usually given in units of Bq/g, butanother commonly used unit of activity is the curie (Ci) allowing thedefinition of specific activity in Ci/g.

The term “alkyl” by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain hydrocarbon,having the number of carbon atoms designated (i.e. C1.6 meaning one tosix carbons). Representative alkyl groups include straight and branchedchain alkyl groups having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms.Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl,n-octyl, and the like. The term “deuteroalkyl” refers to a deuteratedanalog of an alkyl group.

“Optional” or “Optionally” as used throughout the disclosure means thatthe subsequently described event or circumstance may or may not occur,and that the description includes instances where the event orcircumstance occurs and instances in which it does not. For example, thephrase “the alkyl group is optionally substituted with one or twosubstituents” means that the substituent may but need not be present,and the description includes situations where the alkyl group issubstituted and situations where the alkyl group is not substituted.

It will be noted that the structure of some of the compounds of theinvention include asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of thedisclosure, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Enantiomers (R- andS-configurations) are named according to the system developed by R. S.Cahn, C. Ingold, and V. Prelog.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof a compound of the invention wherein the parent compound is modifiedby forming acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines; alkali (basic) or organicsalts of acidic residues such as carboxylic acids; and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. Commonsalt-forming cations in basic salts include but are not limited tosodium Na⁺, potassium K⁺, calcium Ca⁺, magnesium Mg⁺, ammonium NH₄ ⁺,quaternary ammonium NR₄ ⁺, where R can be an alkyl.

For example, such conventional non-toxic acid salts include, but are notlimited to, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic,glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic,hydrochloric, hydroiodide, hydroxymaleic, hydroxynaphthoic, isethionic,lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic,phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic,succinic, sulphamic, sulphanilic, sulphuric, tannic, tartaric, andtoluene sulphonic.

“Solvate”, as used herein, refers to a solvent addition form of acompound of the invention that contains either stoichiometric ornon-stoichiometric amounts of solvent. Some compounds have a tendency totrap a fixed molar ratio of solvent molecules in the crystalline solidstate, thus forming a solvate. If the solvent is water, the solvateformed is a hydrate, and when the solvent is alcohol, the solvate formedis an alcoholate. Hydrates are formed by the combination of one or moremolecules of water with one of the substances in which the water retainsits molecular state as H₂O, such combination being able to form one ormore hydrate.

The term “tautomer” as used herein means compounds produced by thephenomenon wherein a proton of one atom of a molecule shifts to anotheratom. See, Jerry March, Advanced Organic Chemistry: Reactions,Mechanisms and Structures, Fourth Edition, John Wiley & Sons, pages69-74 (1992). The tautomers also refer to one of two or more structuralisomers that exist in equilibrium and are readily converted from oneisomeric form to another. Examples of include keto-enol tautomers, suchas acetone/propen-2-ol, imine-enamine tautomers and the like, ring-chaintautomers, such as glucose/2,3,4,5,6-pentahydroxy-hexanal and the like,the tautomeric forms of heteroaryl groups containing a —N═C(H)—NH-ringatom arrangement, such as pyrazoles, imidazoles, benzimidazoles,triazoles, and tetrazoles. Where the compound contains, for example, aketo or oxime group or an aromatic moiety, tautomeric isomerism(‘tautomerism’) can occur. The compounds described herein may have oneor more tautomers and therefore include various isomers. A person ofordinary skill in the art would recognize that other tautomeric ringatom arrangements are possible. All such isomeric forms of thesecompounds are expressly included in the present disclosure.

The term “isomers” means compounds having identical molecular formulabut differ in the nature or sequence of bonding of their atoms or in thearrangement of their atoms in space. Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers”.“Stereoisomer” and “stereoisomers” refer to compounds that exist indifferent stereoisomeric forms if they possess one or more asymmetriccenters or a double bond with asymmetric substitution and, therefore,can be produced as individual stereoisomers or as mixtures.Stereoisomers include enantiomers and diastereomers. Stereoisomers thatare not mirror images of one another are termed “diastereomers” andthose that are non-superimposable mirror images of each other are termed“enantiomers”. When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture”. Unless otherwise indicated, the descriptionis intended to include individual stereoisomers as well as mixtures. Themethods for the determination of stereochemistry and the separation ofstereoisomers are well-known in the art (see discussion in Chapter 4 ofADVANCED ORGANIC CHEMISTRY, 6th edition J. March, John Wiley and Sons,New York, 2007) differ in the chirality of one or more stereocenters.

The term “prodrugs” as used herein means any compound which releases abiologically active compound or drug in vivo when such prodrug isadministered to a subject. Prodrugs of a compound of Formula I areprepared by modifying functional groups present in the compound ofFormula I in such a way that the modifications may be cleaved in vivo torelease the active compound. Prodrugs may be prepared by modifyingfunctional groups present in the compounds in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe active compounds. Prodrugs include compounds of Formula I wherein ahydroxy, amino, carboxyl or sulfonyl group in a compound of Formula I isbonded to any group that may be cleaved in vivo to regenerate the freehydroxyl, amino, or sulfonate group, respectively. Examples of prodrugsinclude, but are not limited to esters (e.g., acetate, formate, andbenzoate derivatives), amides, guanidines, carbamates (e.g.,N,N-dimethylaminocarbonyl) of hydroxy functional groups in compounds ofFormula I, and the like. Preparation, selection, and use of prodrugs isdiscussed in T. Higuchi and V. Stella, “Pro-drugs as Novel DeliverySystems,” Vol. 14 of the A.C.S. Symposium Series; “Design of Prodrugs”,ed. H. Bundgaard, Elsevier, 1985; and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987, each of which are hereby incorporated by referencein their entirety.

As defined herein, the term “metabolite” can refer to amino acidconjugates, glucuronidated and sulphated derivatives of the compoundsdescribed herein, wherein one or more amino acid, glucuronic acid orsulphate moieties are linked to compound of the invention. Sulphatedderivatives of the compounds may be formed through sulphation of thehydroxyl groups (e.g., 3-hydroxyl, 7-hydroxyl, 11-hydroxyl, and/or thehydroxyl of the R₇ group). Examples of such metabolites include, but arenot limited to 3-sulphate, 7-sulphate, 11-sulphate, 3,7-bisulphate,3,11-bisulphate, 7,11-bisulphate, and 3,7,11-trisulphate of thecompounds described herein.

As used herein, the term “amino acid conjugates” refers to conjugates ofa compound of the invention with any suitable amino acid. Taurine(—NH(CH₂)₂SO₃H), glycine (—NHCH₂CO₂H), and sarcosine (—N(CH₃)CH₂CO₂H)are examples of amino acid conjugates. Suitable amino acid conjugates ofthe compounds have the added advantage of enhanced integrity in bile orintestinal fluids. Suitable amino acids are not limited to taurine,glycine, and sarcosine.

As defined herein, the term “glucuronides” refers to glucuronidatedderivatives of the compounds described herein, wherein one or moreglucuronic acid linked to compound of the disclosure. Glucuronic acidmoieties may be linked to the compounds through glycosidic bonds withthe hydroxyl groups of the compounds (e.g., 3-hydroxyl, 7-hydroxyl,11-hydroxyl, 12-hydroxyl, and/or the hydroxyl of the R¹ group). Examplesof glucuronides of Compound of Formula I include, but are not limitedto, 3-O-glucuronides, 7-O-glucuronides, 12-O-glucuronides,3-O-7-O-diglucuronides, 3-O-12-O-triglucuronides,7-O-12-O-triglucuronides, and 3-O-7-O-12-O-triglucuronides.

As defined herein, the terms “sulphated derivatives” and/or “sulphates”relate to the compounds described herein, wherein one or more sulphatemoieties are linked to compound of the disclosure. Sulphated derivativesof the compounds may be formed through sulphation of the hydroxyl groups(e.g., 3-hydroxyl, 7-hydroxyl, 11-hydroxyl, 12-hydroxyl, and/or thehydroxyl of the R¹ group). Examples of sulphated derivatives of compoundof Formula I include, but are not limited to 3-sulphates, 7-sulphates,11-sulphates, 12-sulphates, 3,7-bisulphates, 3,12-bisulphates,7,12-bisulphates, and 3,7,12-trisulphates and other combinationsthereof.

A “composition” or “pharmaceutical composition” is a formulationcontaining a compound of the invention or a salt, solvate, or amino acidconjugate thereof. In one embodiment, the pharmaceutical composition isin bulk or in unit dosage form. The unit dosage form is any of a varietyof forms, including, for example, a capsule, an IV bag, a tablet, asingle pump on an aerosol inhaler, or a vial. The quantity of activeingredient (e.g., a formulation of a compound of the invention or saltsthereof) in a unit dose of composition is an effective amount and isvaried according to the particular treatment involved. One skilled inthe art will appreciate that it may be necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, ocular, ophthalmic,pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous,intramuscular, intraperitoneal, intranasal, and the like. Dosage formsfor the topical or transdermal administration of a compound of thisapplication include powders, sprays, ointments, pastes, creams, lotions,gels, solutions, patches and inhalants. In another embodiment, theactive compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier is“acceptable” in the sense of being compatible with the other ingredientsof a subject composition and not injurious to the patient. In certainembodiments, a pharmaceutically acceptable carrier is non-pyrogenic.Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The term “treating”, as used herein, refers to relieving, lessening,reducing, eliminating, modulating, or ameliorating, i.e., causingregression of the disease state or condition.

The term “preventing”, as used herein, refers to completely or almostcompletely stop a disease state or condition, from occurring in apatient or subject, especially when the patient or subject ispredisposed to such or at risk of contracting a disease state orcondition. Preventing can also include inhibiting, i.e., arresting thedevelopment, of a disease state or condition, and relieving orameliorating, i.e., causing regression of the disease state orcondition, for example when the disease state or condition may alreadybe present.

The phrase “reducing the risk of”, as used herein, refers to loweringthe likelihood or probability of a central nervous system disease,inflammatory disease and/or metabolic disease from occurring in apatient, especially when the subject is predisposed to such occurrence.

As used herein, the term “modulating” or “modulate” or the like refersto an effect of altering a biological activity, especially a biologicalactivity associated with a particular biomolecule (e.g., receptor orenzyme). For example, an agonist or antagonist of a particularbiomolecule modulates the activity of that biomolecule by eitherincreasing (e.g. agonist, activator), or decreasing (e.g. antagonist,inhibitor) the activity of the biomolecule. Such activity is typicallyindicated in terms of an inhibitory concentration (IC₅₀) or effectiveconcentration (EC₅₀) of the compound for an inhibitor or activator,respectively, with respect to, for example, a receptor or an enzyme.

“Combination therapy” (or “co-therapy”) refers to the administration ofa compound of the invention and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents (i.e., the compound ofthe invention and at least a second agent). The beneficial effect of thecombination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time periods (usually minutes,hours, days or weeks depending upon the combination selected).“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present application. “Combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected may be administered by intravenous injection while the othertherapeutic agents of the combination may be administered orally.Alternatively, for example, all therapeutic agents may be administeredorally or all therapeutic agents may be administered by intravenousinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery ormechanical treatments). Where the combination therapy further comprisesa non-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

An “effective amount” of a compound of the invention, or a combinationof compounds is an amount (quantity or concentration) of compound orcompounds. In one embodiment, when a therapeutically effective amount ofa compound is administered to a subject in need of treatment symptomsarising from the disease are ameliorated immediately or afteradministration of the compound one or more times. The amount of thecompound to be administered to a subject will depend on the specificdisorder, the mode of administration, co-administered compounds, if any,and the characteristics of the subject, such as general health, otherdiseases, age, sex, genotype, body weight, and tolerance to drugs. Theskilled artisan will be able to determine appropriate dosages dependingon these and other factors. For any compound, the therapeuticallyeffective amount can be estimated initially either in cell cultureassays, e.g., of neoplastic cells, or in animal models, usually rats,mice, rabbits, dogs, or pigs. The animal model may also be used todetermine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

The term “prophylactically effective amount” means an amount (quantityor concentration) of a compound of the present invention, or acombination of compounds, that is administered to prevent or reduce therisk of a disease—in other words, an amount needed to provide apreventative or prophylactic effect. Therapeutic/prophylactic efficacyand toxicity may be determined by standard pharmaceutical procedures,e.g., ED₅₀ (the dose therapeutically effective in 50% of the population)and LD₅₀ (the dose lethal to 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index, and itcan be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositionsthat exhibit large therapeutic indices are preferred. The dosage mayvary depending upon various factors, including but not limited to thedosage form employed, sensitivity of the patient, and the route ofadministration.

The amount of the present compound to be administered to a subject willdepend on a particular disorder, mode of administration, co-administeredcompounds, if any, and the characteristics of the subject, such asgeneral health, other diseases, age, sex, genotype, body weight, andtolerance to drugs. The skilled artisan will be able to determineappropriate dosages depending on these and other factors.

A “subject” includes mammals, e.g., humans, companion animals (e.g.,dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs,horses, and the like), and laboratory animals (e.g., rats, mice, guineapigs, and the like). Typically, the subject is human.

The term “assay” or “assaying” relates to the creation of experimentalconditions and the gathering of data regarding a particular result ofthe exposure to specific experimental conditions. For example,biomolecules or target molecules, e.g., enzymes or receptors, can beassayed based on their ability to act upon a detectable substrate. Acompound can be assayed based on its ability to bind to a particulartarget molecule or molecules.

As used herein, the terms “ligand” and “modulator” may be usedequivalently to refer to a compound that changes (i.e., increases ordecreases) the activity of a target biomolecule, e.g., an enzyme orreceptor. Generally, a ligand or modulator is a compound that possessespharmacological and/or pharmacokinetic properties for a particularbiological system or therapeutic use.

The term “binds” in connection with the interaction between a target anda potential binding compound indicates that the potential bindingcompound associates with the target to a statistically significantdegree as compared to association with proteins generally (i.e.,non-specific binding). Thus, the term “binding compound” refers to acompound that has a statistically significant association with a targetmolecule. In some embodiments, a binding compound interacts with aspecified target with a dissociation constant (K_(D)) of about 1 mM orless, about 1 μM or less, e.g., about 500 nm, about 400 nm, about 300nm, about 200 nm, or about 100 nm, about 50 nM or less, about 10 nM orless, or about 1 nM or less. In the context of compounds binding to atarget, the terms “greater affinity” and “selective” indicates that thecompound binds more tightly than a reference compound, or than the samecompound in a reference condition, i.e., with a lower dissociationconstant. In some embodiments, the greater affinity is at least about 2,3, 4, 5, 8, 10, 30, 50, 100, 200, 400, 500, 1000, or 10,000-fold greateraffinity.

Compounds of the Disclosure

The present disclosure relates to isotopically labeled (e.g.,deuterated) derivatives of bile acids (e.g., obeticholic acid) includingamino acid conjugates and glucuronides thereof.

In some of the embodiments, the present disclosure pertains to acompound of Formula I.

or a pharmaceutically acceptable salt thereof, wherein

R₁ is OH, O-glucuronide, OSO₃H, SO₃H, CO₂R₅, ¹⁴CO₂R₅, C(O)R₆, or¹⁴C(O)R₆;

R₂ is H, D, or OH;

R₃ is OH or O-glucuronide;

R₄ is OH or O-glucuronide;

R₅ is H or substituted or unsubstituted alkyl;

R₆ is NH(CH₂)₂SO₃H, NHCH₂CO₂H, or N(CH₃)CH₂CO₂H or glucuronic acidmoieties, wherein hydrogens can be replaced with deuterium;

R₁₁ is Z₁₁ b, hydroxy, halogen, alkoxy, or oxo when Z_(11a) is notpresent;

R₁₆ is Z_(16a), hydroxy, halogen, alkoxy, or oxo when Z_(16b) is notpresent; R₂₃ is Z_(23a) or alkyl; and

Z_(1a), Z_(1b), Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₅, Z₆, Z₇, Z₈, Z₉,Z_(11a), Z_(11b), Z₁₂, Z₁₄, Z_(15a), Z_(15b), Z_(16a), Z_(16b), Z₁₇,Z₁₈, Z₁₉, Z₂₀, Z₂₁, Z_(22a), Z_(22b), Z_(23a), Z_(23b), Z_(24a),Z_(24b), or Z₂₅ is independently selected from H (hydrogen) or D(deuterium), and at least one of Z_(1a), Z_(1b), Z_(2a), Z_(2b), Z₃,Z_(4a), Z_(4b), Z₅, Z₆, Z₇, Z₈, Z₉, Z_(11a), Z_(11b), Z₁₂, Z₁₄, Z_(15a),Z_(15b), Z_(16a), Z_(16b), Z₁₇, Z₁₈, Z₁₉, Z₂₀, Z₂₁, Z_(22a), Z_(22b),Z_(23a), Z_(23b), Z_(24a), Z_(24b), or Z₂₅ is D; and

any carbon atom is ¹¹C, ¹²C, ¹³C, or ¹⁴C, any nitrogen atom is ¹³N, ¹⁴Nor ¹⁵N and any oxygen atom is ¹⁵O, ¹⁶O, ¹⁷O, or ¹⁸O.

In some of the embodiments, R₂ is hydrogen. In some of the embodiments,R₂ is hydroxy. In some of the embodiments, R₂ is alpha-hydroxy.

In some of the embodiments, R₁₁ is hydroxy or oxo.

In some of the embodiments, R₁₆ is hydroxy or oxo.

In some of the embodiments, R₂ is hydrogen and R₁₁ is hydroxy.

In some of the embodiments, R₂ is hydroxy and R₁₁ is hydroxy.

In some embodiments R₂₃ is methyl. In some embodiments R₂₃ is (S-methyl.In some embodiments R₂₃ is (R)-methyl.

In some embodiment, R₆ is taurine (—NH(CH₂)₂SO₃H), glycine (—NHCH₂CO₂H),or sarcosine (—N(CH₃)CH₂CO₂H).

In certain embodiments, the present disclosure relates to compounds ofFormula I having a chemical structure corresponding to Formulas II, III,IV, and V.

In certain embodiments, the present disclosure relates to a compound ofFormula II

wherein

Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) isindependently selected from H or D and at least one of Z_(2a), Z_(2b),Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) is D and R₁, R₂, R₃,and R₄ are as described above.

In some of the embodiments, at least two of Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) are D. In some of theembodiments, at least three of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆,Z₇, Z₈, Z_(23a), or Z_(23b) are D. In some of the embodiments, at leastfour of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), orZ_(23b) are D. In some of the embodiments, at least five of Z_(2a),Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) are D. Insome of the embodiments, at least six of Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) are D. In some of theembodiments, at least seven of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆,Z₇, Z₈, Z_(23a), or Z_(23b) are D. In some of the embodiments, at leasteight of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), orZ_(23b) are D. In some of the embodiments, at least nine of Z_(2a),Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) are D. Insome embodiments, Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈,Z_(23a), and Z_(23b) are D.

In some of the embodiments, R₂ is hydrogen. In some of the embodiments,R₂ is hydroxy. In some of the embodiments, R₂ is alpha-hydroxy.

In some embodiments, any carbon atom of the compound of Formula II, or apharmaceutically acceptable salt thereof, is ¹¹C, ¹²C, ¹³C, or ¹⁴C, anynitrogen atom is ¹³N, ¹⁴N or ¹⁵N, and any oxygen atom is ¹⁵O, ¹⁶O, ¹⁷O,or ¹⁸O.

In certain embodiments, the present disclosure relates to a compound ofFormula III

wherein

Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(11a), Z_(23a), orZ_(23b) is independently selected from H or D and at least one ofZ_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) is Dand R₁, R₂, R₃, R₄, and Ru are as described above.

In some of the embodiments, at least two of Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₆, Z₇, Z₈, Z_(11a), Z_(23a), or Z_(23b) are D. In some of theembodiments, at least three of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆,Z₇, Z₈, Z_(11a), Z_(23a), or Z_(23b) are D. In some of the embodiments,at least four of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈,Z_(11a), Z_(23a), or Z_(23b) are D. In some of the embodiments, at leastfive of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(11a),Z_(23a), or Z_(23b) are D. In some of the embodiments, at least six ofZ_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(11a), Z_(23a), orZ_(23b) are D. In some of the embodiments, at least seven of Z_(2a),Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(11a), Z_(23a), or Z_(23b) areD. In some of the embodiments, at least eight of Z_(2a), Z_(2b), Z₃,Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(11a), Z_(23a), or Z_(23b) are D. In someof the embodiments, at least nine of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b),Z₆, Z₇, Z₈, Z_(11a), Z_(23a), or Z_(23b) are D. In some embodiments, atleast ten Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(11a),Z_(23a), and Z_(23b) are D. In some embodiments, Z_(2a), Z_(2b), Z₃,Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(11a), Z_(23a), and Z_(23b) are D.

In some of the embodiments, R₂ is hydrogen. In some of the embodiments,R₂ is hydroxy. In some of the embodiments, R₂ is alpha-hydroxy.

In some of the embodiments, R₁₁ is hydroxy or oxo.

In some of the embodiments, R₂ is hydrogen and Ru is hydroxy.

In some of the embodiments, R₂ is hydroxy and Ru is hydroxy.

In certain embodiments, the present disclosure relates to a compound ofFormula IV

wherein

Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, or Z_(23b) isindependently selected from H or D and at least one of Z_(2a), Z_(2b),Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, or Z_(23b) is D, R₂₃ is alkyl, and R₂,R₃, R₄, and are as described above.

In some of the embodiments, at least two of Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₆, Z₇, Z₈, or Z_(23b) are D. In some of the embodiments, atleast three of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, orZ_(23b) are D. In some of the embodiments, at least four of Z_(2a),Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, or Z_(23b) are D. In some of theembodiments, at least five of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆,Z₇, Z₈, or Z_(23b) are D. In some of the embodiments, at least six ofZ_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, or Z_(23b) are D. Insome of the embodiments, at least seven of Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₆, Z₇, Z₈, or Z_(23b) are D. In some of the embodiments, atleast eight of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, orZ_(23b) are D. In some of the embodiments, Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₆, Z₇, Z₈, or Z_(23b) are D.

In some of the embodiments, R₂ is hydrogen. In some of the embodiments,R₂ is hydroxy. In some of the embodiments, R₂ is alpha-hydroxy.

In some embodiments R₂₃ is methyl. In some embodiments R₂₃ is(S)-methyl. In some embodiments R₂₃ is (R)-methyl.

In some embodiments, any carbon atom of the compound of Formula IV, or apharmaceutically acceptable salt thereof, is ¹¹C, ¹²C, ¹³C, or ¹⁴C, anynitrogen atom is ¹³N, ¹⁴N or ¹⁵N, and any oxygen atom is ¹⁵O, ¹⁶O, ¹⁷O,or ¹⁸O.

In certain embodiments, the present disclosure relates to a compound ofFormula V

wherein

Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(16b) or Z_(23b) isindependently selected from H or D and at least one of Z_(2a), Z_(2b),Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(16b), Z_(23a) or Z_(23b) is D, R₁₆ ishydroxy, and R₂, R₃, R₄, and R₂₃ are as described above.

In some of the embodiments, at least two of Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₆, Z₇, Z₈, Z_(16b), Z_(23a) or Z_(23b) are D. In some of theembodiments, at least three of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆,Z₇, Z₈, Z_(16b), Z_(23a) or Z_(23b) are D. In some of the embodiments,at least four of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈,Z_(16b), Z_(23a) or Z_(23b) are D. In some of the embodiments, at leastfive of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(16b), Z_(23a)or Z_(23b) are D. In some of the embodiments, at least six of Z_(2a),Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(16b), Z_(23a) or Z_(23b) areD. In some of the embodiments, at least seven of Z_(2a), Z_(2b), Z₃,Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(16b), Z_(23a) or Z_(23b) are D. In someof the embodiments, at least eight of Z_(2a), Z_(2b), Z₃, Z_(4a),Z_(4b), Z₆, Z₇, Z₈, Z_(16b), Z_(23a) or Z_(23b) are D. In some of theembodiments, at least nine of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆,Z₇, Z₈, Z_(16b), Z_(23a) or Z_(23b) are D. In some of the embodiments,at least ten of Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(16b),Z_(23a) or Z_(23b) are D. In some of the embodiments, Z_(2a), Z_(2b),Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(16b), Z_(23a) or Z_(23b) are D.

In some of the embodiments, R₂ is hydrogen. In some of the embodiments,R₂ is hydroxy. In some of the embodiments, R₂ is alpha-hydroxy.

In some embodiments R₁₆ is alpha-hydroxy. In some embodiments R₁₆ isbeta-hydroxy. In some embodiments R₂₃ is methyl. In some embodiments R₂₃is (9-methyl. In some embodiments R₂₃ is (R)-methyl.

In some embodiments, any carbon atom of the compound of Formula V, or apharmaceutically acceptable salt thereof, is ¹¹C, ¹²C, ¹³C, or ¹⁴C, anynitrogen atom is ¹³N, ¹⁴N ¹⁵N, and any oxygen atom is ¹⁵O, ¹⁶O, ¹⁷O, or¹⁸O.

In some embodiments, the present disclosure relates to compounds ofFormula VI

wherein

Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) isindependently selected from H or D and at least one of Z_(2a), Z_(2b),Z₃, Z_(4a), Z_(4b), Z₆, Z₇, Z₈, Z_(23a), or Z_(23b) is D and R₁, R₃, andR₄ are as described above.

In some embodiments, the present disclosure relates to compounds ofFormula VII

wherein

-   -   R₁, R₂, R₃, R₄, R₁₁, R₁₆, and R₂₃ are as described above.

In some embodiments, the present disclosure relates to compounds ofFormula VIII

wherein

R₁ is ¹⁴CO₂R₅ or ¹⁴C(O)R₆ and R₂, R₃, R₄, R₁₁, R₁₆, and R₂₃ are asdescribed above.

Synthesis of Compound of the Disclosure

Some embodiments of the present disclosure relate to a process forpreparing the compound of Formula I.

Compounds of Formula I can be prepared by methods known in the art,e.g., those described in U.S. Pat. Nos. 7,932,244; 8,114,862; 9,238,673;and 9,611,289 and publications WO 2014/066819 and WO 2017/062763, theentire contents of each of which are incorporated herein by reference.

Standard synthetic methods and procedures for the preparation of organicmolecules and functional group transformations and manipulations,including the use of protective groups, can be obtained from therelevant scientific literature or from standard reference textbooks inthe field. Although not limited to any one or several sources,recognized reference textbooks of organic synthesis include: Smith, M.B.; March, J. March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5^(th) ed.; John Wiley & Sons: New York, 2001; andGreene, T. W.; Wuts. P. G. M. Protective Groups in Organic Synthesis,3^(rd); John Wiley & Sons: New York, 1999.

In some embodiments, the present disclosure relates to a process forpreparing the compound of Formula II. In some embodiments, the presentdisclosure relates to a process for preparing the compound of FormulaIII. In some embodiments, the present disclosure relates to a processfor preparing the compound of Formula IV. In some embodiments, thepresent disclosure relates to a process for preparing the compound ofFormula V. In some embodiments, the present disclosure relates to aprocess for preparing the compound of Formula VI. In some embodiments,the present disclosure relates to a process for preparing the compoundof Formula VII. In some embodiments, the present disclosure relates to aprocess for preparing the compound of Formula VIII.

In one aspect, this application pertains to methods for preparingdeuterated compounds of Formula (I). Depending on the desired sites ofdeuteration, in some cases deuterium from D₂O can be exchanged directlyinto finished drug compounds or into reagents that are useful forsynthesizing drug molecules (H. Esaki, et al., Tetrahedron, 2006, 62,10954; H. Esaki, et al., Chem. Eur. J., 2007, 13, 4052). In proticsolution exchangeable protons (e.g., such as those in hydroxyl or aminegroup) exchange protons with the solvent. If D₂O is solvent, deuteronswill be incorporated at these positions. The exchange reaction can befollowed using a variety of methods (e.g. NMR spectroscopy). Since thisexchange is an equilibrium reaction, the molar amount of deuteriumshould be high compared to the exchangeable protons of the substrate.For instance, deuterium is added to a compound in H₂O by diluting theH₂O solution with D₂O (e.g. tenfold). Usually exchange is performed atphysiological pH (7.0-8.0). The H/D exchange reaction can also becatalysed, by acid, base or metal catalysts (e.g., platinum). Thedeuteration pattern of a molecule that has undergone H/D exchange can bemaintained in aprotic environments. Deuterium gas is also a usefulstarting material for incorporating deuterium into molecules. Catalyticdeuteration of olefinic and acetylenic bonds is a rapid route forincorporation of deuterium (H. J. Leis, et al., Curr. Org. Chem., 1998,2, 131). Metal catalysts (i.e., Pd, Pt, and Rh) in the presence ofdeuterium gas can be used to directly exchange deuterium for hydrogen infunctional groups containing hydrocarbons (U.S. Pat. No. 3,966,781). Awide variety of deuterated reagents and synthetic building blocks arecommercially available.

Some embodiments of the present disclosure pertain to methods ofpreparing deuterated compounds of Formula I, II, III, IV, V, VI, VII,and VIII based on the methods known in the art including but not limitdto the procedures shown in Schemes 1-11. The methods shown in Schemes1-11 are based on obeticholic acid (OCA) as starting material, but canbe applied to any bile acid analogs of the present disclosure to preparecompounds of Formulas I-VIII.

In some embodiments, d₁₀-OCA analogs can be prepared according toScheme 1. As shown, d₁₀-OCA analogs can be prepared by first treating3,7-diketo-OCA methyl ester with a base, e.g., sodium methoxide in thepresence of deuterated methanol and concommitant treatment with sodiumdeuteroxide in D₂O, at ambient or elevated temperature, to generate3,7-diketo-(d₈)-OCA. Further treatment with deuterated reducing agentsuch as, for example, NaBD₄ generates d₁₀-OCA. The d₁₀-OCA obtained canbe coupled with various moieties, for example, taurine, glycine, otheramino acids, glucuronic acid or their deuterated counterparts, asdescribed herein, to generate d₁₀-OCA derivatives.

In some embodiments, d₇-OCA analogs are prepared according to Scheme 2.As shown, d₇-OCA analogs can be prepared by treating 3,7-diketo-OCA inD₂O with NaOD at ambient temperature. The reaction is monitored by¹H-NMR until the deuterium exchange is complete and d₅-3,7-diketo-OCA isobtained. Next, the d₅-3,7-diketo-OCA intermediate is treated with NaBD₄to generate d₇-OCA. The d₇-OCA obtained can be coupled with variousmoieties, for example, taurine, glycine, other amino acids, glucuronicacid or their deuterated counterparts, as described herein, to generated₇-OCA derivatives.

In some embodiments, d₅-OCA (C3) analogs can be prepared according toScheme 3. As shown, d₅-OCA (C3) analogs can be prepared by oxidizing OCAto 3-keto-OCA using an oxidizing agent, e.g., catalytic TPAP in thepresence of N-methylmorpholine oxide. In some embodiments, reactionusing an oxidizing agent, e.g., catalytic TPAP in the presence ofN-methylmorpholine oxide, provided 3-keto OCA selectively. Reaction of3-keto-OCA with NaOD in D₂O, followed by treatment of NaBD₄ generatesd₅-OCA (C3). The d₅-OCA obtained can be coupled with various moieties,for example, taurine, glycine, other amino acids, glucuronic acid ortheir deuterated counterparts, as described herein, to generate d₅-OCA(C3) derivatives.

In some embodiments, d₅-OCA (C6) analogs can be prepared according toScheme 4. As shown, d₅-OCA (C6) analogs can be prepared by oxidizing OCAto generate 3,7-diketo-OCA. In some embodiments, oxidation can beperformed using, for example, NaOCl or RuCl3 and NaIO₄. In certainembodiments, oxidation of OCA using RuCl3 and NaIO₄ provides fullconversion to 3,7-diketo OCA. Treatment of 3,7-diketo-OCA with sodiumdeuteroxide in D2O at ambient temperature generates 3,7-diketo-(d₅)-OCA.Further treatment of the 3,7-diketo derivative with NaBH₄ generatesd₅-OCA. The d₅-OCA obtained can be coupled with various moieties, forexample, taurine, glycine, other amino acids, glucuronic acid or theirdeuterated counterparts, as described herein, to generate d₅-OCA (C6)derivatives.

In some embodiments, d₄-OCA analogs (C3(d)-C7(d)-C23(d₂)-OCA) can beprepared according to Scheme 5. As shown, d₄-OCA analogs(C3(d)-C7(d)-C23(d₂)-OCA) can be prepared by treating OCA with HCl inthe presence of methanol to generate the OCA methyl ester (OCA-OMe).Deuterium is introduced into the C23 position by treatement of OCA-OMewith NaOMe in MeOD to generate d₂-OCA-OMe. Ester hydrolysis with NaOD inD₂O generates d₂-OCA, which is subjected to oxidation with catalyticRuCl3 in the presence of NaIO₄ to generate d₂-3,7-diketo-OCA. Thediketone is subjected to reduction using NaBD₄ in the presence of NaODin D₂O to generate d₄-OCA. The d₄-OCA obtained can be coupled withvarious moieties, for example, taurine, glycine, other amino acids,glucuronic acid or their deuterated counterparts, to generate d₄-OCAderivatives.

In some embodiments, C23(d₂)-OCA analogs can be prepared according toScheme 6. As shown, C23(d₂)-OCA analogs can be prepared by treating OCAwith HCl in the presence of methanol to generate the OCA methyl ester(OCA-OMe). Deuterium is introduced into the C23 position by treatementof OCA-OMe with NaOMe in MeOD to generate d₂-OCA-OMe. Ester hydrolysiswith NaOD in D₂O generates d₂-OCA. The d₂-OCA obtained can be coupledwith various moieties, for example, taurine, glycine, other amino acids,glucuronic acid or their deuterated counterparts, to generate d₂-OCAderivatives.

In some embodiments, the present disclosure relates to OCA conjugates,where hydrogens of the amino acid residue are fully or partiallysubstituted with deuterium. In one of the embodiment the conjugate isfully deuterated. Any bile acid intermediate prepared by the methodsdescribed herein, including but not limited to procedures shown inSchemes 1-6, can be converted into the corresponding conjugate by usinga suitable coupling reagent. Suitable coupling reagents include, but arenot limited to carbodiimides, for example, dicyclohexylcarbodiimide(DCC), diisopropylcarbodiimide (DIC),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide.HCl (EDAC.HCl, EDC.HCl,WSC.HCl); the phosphonium- and the aminium-(imonium-) type reagentswhich include, for example,benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate (BOP),Benzotriazol-1-yloxy-tripyrrolidinophosphonium hexafluorophosphate(PyBOP®), Bromo-tripyrrolidino-phosphonium hexafluorophosphate(PyBrOP®), 7-aza-benzotriazol-1-yloxy-tripyrrolidinophosphoniumhexafluorophosphate (PyAOP), ethylcyano(hydroxyimino)acetate-02)-tri-(1-pyrrolidinyl)-phosphoniumhexafluorophosphate (PyOxim),3-(diethoxy-phosphoryloxy)-1,2,3-benzo[d]triazin-4(3H)-one (DEPBT),N-[(7-aza-1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-methylmethanaminiumhexafluorophosphate N-oxide (HATU),N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uroniumhexafluorophosphate N-oxide (HDMC),N-[(7-aza-1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-methylmethanaminiumtetrafluoroborate N-oxide (TATU),2-(6-chloro-1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethylaminiumhexafluorophosphate (HCTU),N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uroniumhexafluorophosphate N-oxide (HDMC),2-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethylaminiumtetrafluoroborate/hexafluorophosphate (TBTU (BF₄ ⁻)/HBTU (PF₆ ⁻)),1-[1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino]-uroniumhexafluorophosphate (COMU), tetramethylfluoroformamidiniumhexafluorophosphate (TFFH), and2-(1-oxy-pyridin-2-yl)-1,1,3,3-tetramethylisothiouroniumtetrafluoroborate (TOTT); and other coupling reagents, for example,N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ);2-propanephosphonic acid anhydride (T3P),4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salts (DMTMM)and related compounds, bis-trichloromethylcarbonate or “Triphosgene”(BTC), and 1,1′-Carbonyldiimidazole (CDI).

Some coupling reactions (e.g., amide bond formations) require thepresence of an additive (e.g., to enhance the reactivity and also toreduce formation of epimers as well as N-acylureas), such as, forexample, 1-hydroxybenzotriazole (HOBt), HOBt-6-sulfonamidomethyl resinHCl (200-400 mesh), N-hydroxysuccinimide (HOSu),1-hydroxy-7-aza-1H-benzotriazole (HOAt), ethyl2-cyano-2-(hydroximino)acetate (Oxyma Pure®), and4-(N,N-dimethylamino)-pyridine (DMAP).

Some coupling reactions (e.g., amide bond formations) require thepresence of a base, for example, triethylamine, diisopropylethylamine(DIPEA), N-methylmorpholine (NMM), sym-collidine, 2,6-lutidine, Cs₂CO₃,NaHCO₃, etc.

In some of the embodiments, the present disclosure pertains to a methodof making amino acid conjugates of Formula I.

wherein A is an amino acid residue (protected or unprotected) or anamino acid residue where at least one hydrogen is repaced with deuteriumor tritium (the carboxylic group of amino residue can be protected orunprotected).

In some of the embodiments, glycine conjugate, e.g., d₅-OCA-O (d₂)Gly,can be prepared as shown in Scheme 7.

In some embodiments, d₅-OCA-O (d₂)Gly analogs can be prepared byoxidizing OCA to 3-keto-OCA using, for example, catalytic TPAP in thepresence of N-methylmorpholine oxide. Treating 3-keto-OCA with NaOD inD₂O generates d₄-3-keto-OCA, which is treated with a reducing agent,e.g., NaBD₄ to generate d₅-OCA (as also shown in Schemes 3 and 4).Coupling of d₅-OCA with d₂-glycine methyl ester is achieved using acoupling reagent, e.g.,4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMT-MM), toafford d₅-OCA-O(d₂)Gly-OMe. Ester hydrolysis using NaOD in D₂O affordsd₅-OCA-O (d₂)Gly.

In some of the embodiments tauro conjugate, e.g., d₅-OCA-O(d₄)Tau, canbe prepared as shown in Scheme 8.

In some embodiments, d₅-OCA-O(d₄)Tau analogs can be prepared by treatingd₅-OCA in the presence of d₄-taurine and a coupling reagent (e.g.,N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ),1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (HATU) and 2,6-dichlorobenzoyl chloride, orother peptide coupling agent) and a base (e.g., triethylamine, DIPEA,2,6-lutidine, Cs₂CO₃, etc.).

In some embodiments, as shown, for example, in Schemes 1-6, deuteriumexchange and the reduction (using, e.g., NaBD₄) are carried out in onepot. In some embodiment, deuterium exchange and the reduction arecarried out as two sequential steps.

Some embodiments of the present disclosure relate to compounds ofFormulas I, II, III, IV, V, and VI having at least one carbon-12 (¹²C)replaced with radioactive carbon-14 (¹⁴C) or carbon-13 (¹³C). In certainembodiments, compounds of the present disclosure (Formulas I-VI) arelabeled with at least one ¹⁴C (e.g., compound VII or VIII). Compoundslabeled with ¹⁴C can be prepared by various methods known in the art.

The general approach to quantify a metabolite is to synthesize acarbon-14-labeled version of the drug. By replacing a carbon-12 atomwith radioactive carbon-14, researchers have a chemically identicalanalogue that enables the pathway of the drug to be traced in abiological system. Carbon-14 radioisotopes sometimes are selected overtritium because the exact position of the label can be selected based onthe synthetic route employed for labeling. Carbon occurs in the skeletonof nearly all drug molecules, thereby allowing a chosen position for theradiolabeling site that is more likely to be metabolically stable.

Carbon-14-labeled compounds generally exhibit greater radiochemicalstability than their tritium-labeled counterparts, as a result of thehigher specific activity of tritium-labeled material. This has theeffect of increasing the risk of significant autoradiolysis,(radiochemical decomposition), during storage or usage of theradiolabeled compound. Carbon-14 is also detectable at very low levelsusing scintillation counting, making it useful for studies in whichdoses that run close to the pharmacological threshold are common.

In some of the embodiments, compounds of the present disclosure(Formulas I-VI) can be labeled with at least one ¹⁴C (e.g., compound VIIor VIII). In some embodiments, as shown in Scheme 9, ¹⁴C can beincorporated into the molecule by using a radiolabeled reagent, e.g.,[1-¹⁴C] acetaldehyde.

In some embodiments, starting material, for example, compound C1, in asuitable solvent (e.g., THF) at about −65° C. to about −78° C., e.g.,about −70° C., is treated with LDA in the presence ofchlorotrimethylsilane or other suitable reagent for protection ofC3-hydroxy (or any other hydroxy groups) to prepare compound C2.Compound C2 can be treated with [1-¹⁴C] acetaldehyde and borontrifluoride etherate, in a suitable solvent, e.g., dichloromethane, atabout −65° C. to about −78° C., e.g., −70° C., under inert atmosphere,e.g., nitrogen. To provide compound [¹⁴C] C3. Compound [¹⁴C]C3 can betreated with a base, e.g., sodium hydroxide, in a suitable solvent,e.g., methanol, at about 35° C. to about 55° C., e.g., 45° C. to affordcompound [¹⁴C]C4. Compound [¹⁴C]C4 can be hydrogenated in the presenceof sodium hydroxide, and 5% palladium on carbon at room temperature(e.g., at about 20° C., or about 25° C., or about 30° C.) and then atabout 100° C. to about 130° C., e.g., 115° C. to [¹⁴C]OCA ketone C5.[¹⁴C]OCA ketone C5 can be purified (e.g., by HPLC). In some embodiments,the quality of the radiolabelled precursor [¹⁴C]OCA ketone C5 isconsidered a critical control to the quality of [¹⁴C]OCA C6 compound.Purified [¹⁴C]OCA ketone C5 can be treated with a base, e.g., sodiumhydroxide at about 75° C. to about 85° C., e.g., 80° C. and then aquoussodium borohydride at approximately 100° C. to provide crude [¹⁴C]OCAC6. Crude [¹⁴C]OCA C6 can be purified (e.g., by column chromatography).In some embodiments, [¹⁴C]OCA C6 material from the flash column can betreated with QuadraPure™ Tu palladium scavenger. In some embodiments,the filtered material can be further purified using preparative HPLC(e.g. using C18 column).

In some embodiments, [¹⁴C]OCA is at high specific activity. In one ofembodiments, [¹⁴C]OCA high specific activity component can be combinedwith the OCA cold component at a ratio of [¹⁴C]OCA to OCA to produce[¹⁴C]OCA drug substance with the specific activity required for use inthe [¹⁴C]-OCA solution for administration (e.g., intravenous or oral) inclinical trial or other studies. The material can then be furtherdiluted with OCA to produce [¹⁴C]OCA drug substance with the specificactivity required for use in the [¹⁴C]-OCA Drug in Capsule in clinicaltrial or other study.

In some embodiments, e.g., as shown in Schemes 10 and 11, ¹⁴C can beincorporated into the molecule (compound of Formula I) at position C24by using a radiolabeled reagent, e.g., K¹⁴CN. The methods shown inSchemes 10 or 11 are based on obeticholic acid (OCA) and compound 100,respectively, as starting materials, but can be applied to any bile acidanalogs of the present disclosure (i.e., compound of Formula I) toprepare ¹⁴C derivatives of compounds of Formulas I-VIII.

In some embodiments compounds of Formulas I, II, III, IV, V, VI, VII andVIII are glucuronides. Glucuronides of the present disclosure areprepared via methods known in the art, including Koenigs-Knorr reactions(Anne Wadouachi, et. al., Molecules 16 (2011), 3933-3968). Glucuronatedcompounds of Formula I include, but are not limited to 3-O-glucuronides,7-O-glucuronides, 11-O-glucuronides, 12-O-glucuronides,24-O-glucuronides, or di- or triglucuronides, such as, for example,3,7-diglucuronides or 3,7,11-triglucuronides. Glucuronides of Formula Ican be prepared by methods known in the art and exemplified in Schemes12 and 13.

In some embodiments, the present disclosure relates to 3-O-glucuronidesof Formula I. The methods shown in Scheme 12 are based on obeticholicacid (OCA) as starting material, but can be applied to any bile acidanalogs of the present disclosure (i.e., compound of Formula I) toprepare 3-O-glucuronidated compounds of Formulas I-VIII.

In some embodiments, the stating 3-hydroxy compound (e.g., OCA) can beprotected, e.g., converted into methyl ester using, for example,methanol and para-toluene sulfonic acid. Protected compound, e.g.,OCA-methyl ester, can be treated with silver carbonate andacetobromo-α-D-glucuronic acid methyl ester (protected glucuronic acid)to provide protected 3-O-glucuronide, e.g., protectedOCA-3-O-glucuronide, which can be deprotected using methods known in theart to give 3-O-glucuronide of Formula I, e.g., OCA-3-O-glucuronide. Insome embodiments, glucuronide starting material can be radiolabeled.

In some of the embodiments of the present disclosure, compounds ofFormula I can be tritated. Tritiated compounds of Formula I can beprepared according to methods known in the art, for example, fromcorresponding 3-, 7-, 11-, or 12-keto compounds using tritiated reducingagent such as NaBT₄. In one of the embodiments, compounds of Formula Ihave tritium incorporated at C3 position. In one of the embodiments,compounds of Formula I have tritium incorporated at C11 position. In oneof the embodiments, compounds of Formula I have tritium incorporated atC12 position. In one of the embodiments, tritium can be incorporated atC7 position.

In some embodiments, the present disclosure relates to 24-O-glucuronidesof Formula I. The method shown in Scheme 13 is based on obeticholic acid(OCA) as starting material, but can be applied to any bile acid analogsof the present disclosure to prepare 24-O-glucuronidated compounds ofFormulas I-VIII.

As shown in Scheme 13, C7-tritiated compound of Formula I, e.g.,[³H]OCA, can be prepred from C7-keto compound using tritiated reducingagent (e.g., NaBT₄). Radiolabeled and glucuronated compounds of FormulaI, e.g., [³H]OCA-24-Glucuronide of OCA, can be prepared by methods knownin the art and described herein.

In some embodiments, compound of Formula I having carboxylic or hydroxygroup(s), e.g., at C23 position, can be treated with protectedglucuronic acid, e.g., acetobromo-α-D-glucuronic acid methyl ester, inthe presence of silver carbonate to provide glucuronated compound, e.g.,Intermediate 2. Intermdiate 2 can be purified or used in the next stepwithout purification. Enzyme-catalyzed consecutive deprotection(hydrolysis) affords glucuronated compound of Formula I, e.g.,[³H]OCA-24-glucuronide. Hydrolytic enzymes that can be used fordeprotection include, but are not limited to transferases, hydrolases,and lyases. In some embodiments, deprotection may involve LAS enzyme,CSR enzyme, Cal-B enzyme and combinations thereof.

In some embodiments, the compound of Formula I-VIII can be purifiedusing various methods including column chromatography (e.g., reversedphase chromatography). All radiolabeled compounds of the presentdisclosure can be characterised by various analytical method including,for example, mass spectrometry, HPLC and NMR (¹H, ¹³C, HMBC

(Heteronuclear Multiple Bond Correlation)). In some embodiments, thecompounds of Formulas I-VIII are analysed by NMR spectroscopy. Hydrogenand deuterium nuclei are different in their magnetic properties,therefore it is possible to distinguish between them by NMRspectroscopy. Deuterons will not be observed in a ¹H NMR spectrum andconversely, protons will not be observed in a²H NMR spectrum. Wheresmall signals are observed in a ¹H NMR spectrum of a highly deuteratedsample, these are referred to as residual signals. They can be used tocalculate the level of deuteration in a molecule. Analogous signals arenot observed in ²H NMR spectra because of the low sensitivity of thistechnique compared to the ¹H analysis. Deuterons typically exhibit verysimilar chemical shifts to their analogous protons. Analysis via ¹³C NMRspectroscopy is also possible: the different spin values of hydrogen (½)and deuterium (1) gives rise to different splitting multiplicities. NMRspectroscopy can be used to determine site-specific deuteration ofmolecules. In some embodiments, deuterium incoroporation is determinedby ¹H-NMR. In one of the embodiments, the deuterium incorporation ateach deuterium atom in compounds of the present disclosure is at leastabout 52.5% (enrichment factor is at least about 3500). In one of theembodiments, the deuterium incorporation at each deuterium atom incompounds of the present disclosure is at least about 60% (enrichmentfactor is at least about 4000). In one of the embodiments, the deuteriumincorporation at each deuterium atom in compounds of the presentdisclosure is at least about 67.5% (enrichment factor is at least about4500). In one of the embodiments, the deuterium incorporation at eachdeuterium atom in compounds of the present disclosure is at least about75% (enrichment factor is at least about 5000). In one of theembodiments, the deuterium incorporation in compounds of the presentdisclosure is at least about 82.5% (enrichment factor is at least about5500). In one of the embodiments, the deuterium incorporation incompounds of the present disclosure is at least about 90% (enrichmentfactor is at least about 6000). In one of the embodiments, the deuteriumincorporation in compounds of the present disclosure is at least about95% (enrichment factor is at least about 6333.3). In one of theembodiments, the deuterium incorporation in compounds of the presentdisclosure is at least about 97% (enrichment factor is at least about6466.7). In one of the embodiments, the deuterium incorporation incompounds of the present disclosure is at least about 99% (enrichmentfactor is at least about 6600). In one of the embodiments, the deuteriumincorporation in compounds of the present disclosure is at least about99.5% (enrichment factor is at least about 6633.3).

The exemplified schemes and conditions are not intended to be limiting.

Pharmaceutical Compositions

A “pharmaceutical composition” is a formulation containing an activeagent (e.g., isotopically-labeled compound of Formula I-VIII or apharmaceutically acceptable salt thereof) in a form suitable foradministration to a subject.

In some embodiments, the present disclosure pertains to a pharmaceuticalcomposition comprising the compounds of Formulas I, II, III, IV, V, VI,VII or VIII and a pharmaceutically acceptable diluent, excipient orcarrier. In some embodiments, the present disclosure pertains to apharmaceutical composition comprising the compounds of Formula I and apharmaceutically acceptable diluent, excipient or carrier. In someembodiments, the present disclosure pertains to a pharmaceuticalcomposition comprising the compounds of Formula II and apharmaceutically acceptable diluent, excipient or carrier. In someembodiments, the present disclosure pertains to a pharmaceuticalcomposition comprising the compounds of Formula III and apharmaceutically acceptable diluent, excipient or carrier. In someembodiments, the present disclosure pertains to a pharmaceuticalcomposition comprising the compounds of Formula IV and apharmaceutically acceptable diluent, excipient or carrier. In someembodiments, the present disclosure pertains to a pharmaceuticalcomposition comprising the compounds of Formula V and a pharmaceuticallyacceptable diluent, excipient or carrier. In some embodiments, thepresent disclosure pertains to a pharmaceutical composition comprisingthe compounds of Formula VI and a pharmaceutically acceptable diluent,excipient or carrier. In some embodiments, the present disclosurepertains to a pharmaceutical composition comprising the compounds ofFormula VII and a pharmaceutically acceptable diluent, excipient orcarrier. In some embodiments, the present disclosure pertains to apharmaceutical composition comprising the compounds of Formula VIII anda pharmaceutically acceptable diluent, excipient or carrier.

In one embodiment, the pharmaceutical composition is in bulk or in unitdosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler, or a vial. The quantity of active ingredient in aunit dose of composition is an effective amount and is varied accordingto the particular treatment involved.

The present application provides pharmaceutical compositions comprisinga compound of Formula I or a pharmaceutically acceptable salt thereofand a pharmaceutically acceptable diluent, excipient, or carrier. Thepharmaceutical composition of the present disclosure can be administeredenternally, orally, transdermally, pulmonarily, inhalationally,buccally, sublingually, intraperintoneally, subcutaneously,intramuscularly, intravenously, rectally, intrapleurally, intrathecally,intranasally, parenterally, or topically.

In particular, tablets, coated tablets, capsules, syrups, suspensions,drops or suppositories are used for enteral administration, solutions,preferably oily or aqueous solutions, furthermore suspensions, emulsionsor implants, are used for parenteral administration, and ointments,creams or powders are used for topical application. Suitable dosageforms include, but are not limited to capsules, tablets, pellets,dragees, semi-solids, powders, granules, suppositories, ointments,creams, lotions, inhalants, injections, cataplasms, gels, tapes, eyedrops, solution, syrups, aerosols, suspension, emulsion, which can beproduced according to methods known in the art, for example as describedbelow:

tablets: mixing of active ingredient/sand auxiliaries, compression ofsaid mixture into tablets (direct compression), optionally granulationof part of mixture before compression.

capsules: mixing of active ingredient/s and auxiliaries to obtain aflowable powder, optionally granulating powder, fillingpowders/granulate into opened capsules, capping of capsules.

semi-solids (ointments, gels, creams): dissolving/dispersing activeingredient/s in an aqueous or fatty carrier; subsequent mixing ofaqueous/fatty phase with complementary fatty/aqueous phase,homogenization (creams only).

suppositories (rectal and vaginal): dissolving/dispersing activeingredient/sin carrier material liquified by heat (rectal: carriermaterial normally a wax; vaginal: carrier normally a heated solution ofa gelling agent), casting said mixture into suppository forms, annealingand withdrawal suppositories from the forms.

aerosols: dispersing/dissolving active agents in a propellant, bottlingsaid mixture into an atomizer.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in watersoluble form, for example,water-soluble salts and alkaline solutions. In addition, suspensions ofthe active compounds as appropriate oily injection suspensions may beadministered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, ethyl oleate or triglycerides or polyethylene glycol-400 (thecompounds are soluble in PEG-400). Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension,including, for example, sodium carboxymethyl cellulose, sorbitol, and/ordextran, optionally, the suspension may also contain stabilizers. Foradministration as an inhalation spray, it is possible to use sprays inwhich the active ingredient is either dissolved or suspended in apropellant gas or propellant gas mixture (for example CO₂ orchlorofluorocarbons). The active ingredient is advantageously used herein micronized form, in which case one or more additional physiologicallyacceptable solvents may be present, for example ethanol. Inhalationsolutions can be administered with the aid of conventional inhalers. Inaddition, stabilizers may be added.

Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates, and agents for the adjustment oftonicity such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Dosage forms for the topical or transdermal administration include butare not limited to powders, sprays, ointments, pastes, creams, lotions,gels, solutions, patches and inhalants. In one embodiment, the activeingredient is mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers or propellantsthat are required.

Suitable excipients are organic or inorganic substances, which aresuitable for enteral (for example oral), parenteral or topicaladministration and do not react with the products of the disclosure, forexample water, vegetable oils, benzyl alcohols, alkylene glycols,polyethylene glycols, glycerol triacetate, gelatine, carbohydrates, suchas lactose, sucrose, mannitol, sorbitol or starch (maize starch, wheatstarch, rice starch, potato starch), cellulose preparations and/orcalcium phosphates, for example tricalcium phosphate or calcium hydrogenphosphate, magnesium stearate, talc, gelatine, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose,polyvinyl pyrrolidone and/or vaseline. If desired, disintegrating agentsmay be added such as the above-mentioned starches and alsocarboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, oralginic acid or a salt thereof, such as sodium alginate. Auxiliariesinclude, without limitation, flow-regulating agents and lubricants, forexample, silica, talc, stearic acid or salts thereof, such as magnesiumstearate or calcium stearate, and/or polyethylene glycol.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin. Sterileinjectable solutions can be prepared by incorporating the activeingredient in the required amount in an appropriate solvent with one ora combination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active ingredient into a sterile vehicle that containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. The compounds of the disclosure can be used, forexample, for the production of injection preparations. The preparationsindicated can be sterilized and/or can contain excipients such aslubricants, preservatives, stabilizers and/or wetting agents,emulsifiers, salts for affecting the osmotic pressure, buffersubstances, colorants, flavourings and/or aromatizers. They can, ifdesired, also contain one or more further active compounds, e.g. one ormore vitamins.

For administration by inhalation, the active ingredient is delivered inthe form of an aerosol spray from pressured container or dispenser,which contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active ingredient is formulated intoointments, salves, gels, or creams as generally known in the art.

One skilled in the art will appreciate that it is sometimes necessary tomake routine variations to the dosage depending on, for example, the ageand condition of the patient. The dosage will also depend on the routeof administration.

One skilled in the art will recognize the advantages of certain routesof administration. The dosage administered will be dependent upon theage, health, and weight of recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired.

In one embodiment, the pharmaceutical composition of the presentapplication is administered orally.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active ingredient can be incorporated withexcipients and used in the form of tablets, troches, or capsules. Oralcompositions can also be prepared using a fluid carrier for use as amouthwash, wherein the compound in the fluid carrier is applied orallyand swished and expectorated or swallowed. Pharmaceutically compatiblebinding agents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring. Forexample, oral compositions can be tablets or gelatin capsules comprisingthe active ingredient together with a) diluents, e.g., lactose,dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b)lubricants, e.g., silica, talcum, stearic acid, its magnesium or calciumsalt and/or polyethyleneglycol; for tablets also c) binders, e.g.,magnesium aluminum silicate, starch paste, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose and orpolyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar,alginic acid or its sodium salt, or effervescent mixtures; and/or e)absorbents, colorants, flavors and sweeteners.

Dragee cores are provided with suitable coatings, which, if desired, areresistant to gastric juices. For this purpose, concentrated saccharidesolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide,lacquer solutions and suitable organic solvents or solvent mixtures.

In order to produce dosage form coatings resistant to gastric juices orto provide a dosage form affording the advantage of prolonged action(modified release dosage form), the tablet, dragee or pill can comprisean inner dosage and an outer dosage component me latter being in theform of an envelope over the former. The two components can be separatedby an enteric layer, which serves to resist disintegration in thestomach and permits the inner component to pass intact into the duodenumor to be delayed in release. A variety of materials can be used for suchenteric layers or coatings, such materials including a number ofpolymeric acids and mixtures of polymeric acids with such materials asshellac, acetyl alcohol, solutions of suitable cellulose preparationssuch as acetyl-cellulose phthalate, cellulose acetate orhydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs orpigments may be added to the tablets or dragee coatings, for example,for identification or in order to characterize combinations of activecompound doses. Suitable carrier substances are organic or inorganicsubstances which are suitable for enteral (e.g. oral) or parenteraladministration or topical application and do not react with thecompounds of disclosure, for example water, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactoseor starch, magnesium stearate, talc and petroleum jelly.

Other pharmaceutical preparations, which can be used orally includepush-fit capsules made of gelatine, as well as soft, sealed capsulesmade of gelatine and a plasticizer such as glycerol or sorbitol. Thepush-fit capsules can contain the active compounds in the form ofgranules, which may be mixed with fillers such as lactose, binders suchas starches, and/or lubricants such as talc or magnesium stearate and,optionally, stabilizers. In soft capsules, the active compounds arepreferably dissolved or suspended in suitable liquids, such as fattyoils, or liquid paraffin.

The liquid forms in which the compositions of the present disclosure maybe incorporated for administration orally include aqueous solutions,suitably flavoured syrups, aqueous or oil suspensions, and flavouredemulsions with edible oils such as cottonseed oil, sesame oil, coconutoil or peanut oil, as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic and natural gums such as tragacanth,acacia, alginate, dextran, sodium carboxymethylcellulose,methylcellulose, polyvinyl-pyrrolidone or gelatine.

Dosage forms for oral administraton can comprise modified releaseformaulations. The term “immediate release” is defined as a release of acompound of Formula I or a pharmaceutically acceptable salt thereof froma dosage form in a relatively brief period of time, generally up toabout 60 minutes. The term “modified release” is defined to includedelayed release, extended release, and pulsed release. The term “pulsedrelease” is defined as a series of releases of drug from a dosage form.The term “sustained release” or “extended release” is defined ascontinuous release of a compound of Formula I or a pharmaceuticallyacceptable salt thereof from a dosage form over a prolonged period oftime.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activeingredient calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the application are dictated by anddirectly dependent on the unique characteristics of the activeingredient and the particular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the application vary depending onthe agent, the age, weight, and clinical condition of the recipientpatient, and the experience and judgment of the clinician orpractitioner administering the therapy, among other factors affectingthe selected dosage.

Dosages of the compounds of the disclosure can range from about 0.01mg/kg per day to about 500 mg/kg per day. In one of the embodiments, thedaily dose is preferably between about 0.01 mg/kg and 10 mg/kg of bodyweight.

In one of the embodiments, the composition or formulation comprisesabout 0.1 mg to about 1500 mg of the compound of Formula I or apharmaceutically acceptable salt thereof per dosage form. In anotherembodiment, the formulation or composition comprises about 1 mg to about100 mg of the compound of Formula I or a pharmaceutically acceptablesalt thereof. In another embodiment, the formulation comprises about 1mg to about 50 mg. In another embodiment, the formulation comprisesabout 1 mg to about 30 mg. In another embodiment, the formulationcomprises about 4 mg to about 26 mg. In another embodiment, theformulation comprises about 5 mg to about 25 mg. In one embodiment, theformulation comprises about 1 mg to about 5 mg. In one embodiment, theformulation comprises about 1 mg to about 2 mg.

An effective amount of a pharmaceutical agent is that which provides anobjectively identifiable improvement as noted by the clinician or otherqualified observer.

The pharmaceutical compositions can be included in a container, kit,pack, or dispenser together with instructions for administration.

The pharmaceutical compositions containing free form, salts, and/orsolid state forms thereof of the present application may be manufacturedin a manner that is generally known, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, or lyophilizing processes. Pharmaceuticalcompositions may be formulated in a conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients and/orauxiliaries that facilitate processing of the active ingredient intopreparations that can be used pharmaceutically. Of course, theappropriate formulation is dependent upon the route of administrationchosen.

Techniques for formulation and administration of the compounds ofFormula I or the pharmaceutically acceptable salts thereof can be foundin Remington: The Science and Practice of Pharmacy, 19^(th) edition,Mack Publishing Co., Easton, Pa. (1995) or any later versions thereof.

The active ingredient can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

Methods for preparation of such formulations will be apparent to thoseskilled in the art.

Methods of the Application

Some aspects of the present disclosure pertain to a method of treating,preventing, ameliorating or modulating a varierty of liver, metabolic,kidney, cardiovascular, gastrointestinal and cancerous diseases,disorders or conditions using deuterated and/or radiolabeled bile acidderivatives of Formula I (including compounds of Formula II, III, IV, V,VI, and VII). In some embodiments, the compounds of Formula I orpharmaceutically acceptable salts thereof are used to improve metabolicprofile, safety, tolerability and/or efficacy.

In some of the embodiments, this application pertains to a method ofmodulating FXR (e.g., activating FXR) in a subject in need thereof,comprising administering a therapeutically effective amount of acompound of Formula I or a pharmaceutically acceptable salt thereof.

In some embodiments, the present disclosure relates to a method oftreating, preventing or ameliorating an FXR-mediated disease or disorderin a subject in need thereof, comprising administering a therapeuticallyeffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt thereof.

In some of the embodiments, this application pertains to a method ofmodulating TGR5 (e.g., activating TGR5) in a subject in need thereof,comprising administering a therapeutically effective amount of acompound of Formula I or a pharmaceutically acceptable salt thereof.

In some aspects, the present disclosure pertains to a compound ofFormula I or a pharmaceutically acceptable salt thereof for treating orpreventing or ameliorting an TGR5-mediated disease or disorder.

In certain embodiments, this disclosure pertains to a method of treatingor preventing an FXR- or TGR5-mediated condition, disease or disorder ina subject in need thereof, comprising administering a compositioncomprising a therapeutically effective amount of a compound of Formula Ior a pharmaceutically acceptable salt thereof.

In one embodiment, the disclosure relates to a method of treating orpreventing chronic liver disease in a subject, comprising administeringto the subject in need thereof an effective amount of a compound ofFormula I or a pharmaceutically acceptable salt thereof. In oneembodiment, the disclosure relates to a method of treating chronic liverdisease. In one embodiment, the disclosure relates to a method ofpreventing chronic liver disease. In one embodiment, the FXR mediatedliver disease is selected from a cholestatic liver disease such asprimary biliary cirrhosis (PBC) also known as primary biliarycholangitis (PBC), primary sclerosing cholangitis (PSC), chronic liverdisease, nonalcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), hepatitis C infection, alcoholic liver disease,liver damage due to progressive fibrosis, and liver fibrosis. Otherexamples of FXR mediated diseases also include portal hypertension, bileacid diarrhea, hyperlipidemia, high LDL-cholesterol, high HDLcholesterol, high triglycerides, and cardiovascular disease. Other liverdiseases include cerebrotendinous xanthomatosis (CTX), drug inducedcholestasis, intrahepatic cholestasis of pregnancy, parenteral nutritionassociated cholestasis (PNAC), bacterial overgrowth or sepsis associatedcholestasis, autoimmune hepatitis, chronic viral hepatitis, livertransplant associated graft versus host disease, living donor transplantliver regeneration, congenital hepatic fibrosis, choledocholithiasis,granulomatous liver disease, intra- or extrahepatic malignancy,Sjogren's syndrome, Sarcoidosis, Wilson's disease, Gaucher's disease,hemochromatosis, and alpha 1-antitrypsin deficiency.

In one embodiment, the disclosure relates to a method of treating orpreventing one or more symptoms of cholestasis, including complicationsof cholestasis in a subject, comprising administering to the subject inneed thereof an effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof. In one embodiment, thedisclosure relates to a method of modulating one or more symptoms ofcholestasis.

A compound of Formula I or a pharmaceutically acceptable salt thereofmay be used for treating or preventing one or more symptoms ofintrahepatic or extrahepatic cholestasis, including without limitation,biliary atresia, obstetric cholestasis, neonatal cholestasis, druginduced cholestasis, cholestasis arising from Hepatitis C infection,chronic cholestatic liver disease such as primary biliary cirrhosis(PBC), and primary sclerosing cholangitis (PSC).

In one embodiment, the present disclosure relates to a method ofenhancing liver regeneration in a subject, comprising administering tothe subject in need thereof an effective amount of a compound of FormulaI or a pharmaceutically acceptable salt thereof. In one embodiment, themethod is enhancing liver regeneration for liver transplantation.

In one embodiment, the disclosure relates to a method of treating orpreventing fibrosis in a subject, comprising administering to thesubject in need thereof an effective amount of a compound of Formula Ior a pharmaceutically acceptable salt thereof.

Accordingly, as used herein, the term fibrosis refers to all recognizedfibrotic disorders, including fibrosis due to pathological conditions ordiseases, fibrosis due to physical trauma (“traumatic fibrosis”),fibrosis due to radiation damage, and fibrosis due to exposure tochemotherapeutics. As used herein, the term “organ fibrosis” includesbut is not limited to liver fibrosis, fibrosis of the kidneys, fibrosisof lung, and fibrosis of the intestine.

In one of the embodiments, the renal disease is diabetic nephropathy,focal segmental glomerulosclerosis (FSGS), hypertensive nephrosclerosis,chronic glomerulonephritis, chronic transplant glomerulopathy, chronicinterstitial nephritis, or polycystic kidney disease.

In some embodiments, the disclosure relates to a method of treating,preventing or modulating cardiovascular disease in a subject, comprisingadministering to the subject in need thereof an effective amount of acompound of Formula I or a pharmaceutically acceptable salt thereof. Inone embodiment, cardiovascular disease is selected from atherosclerosis,arteriosclerosis, dyslipidemia, hypercholesteremia, hyperlipidemia,hyperlipoproteinemia, and hypertriglyceridemia.

The term “hyperlipidemia” refers to the presence of an abnormallyelevated level of lipids in the blood. Hyperlipidemia can appear in atleast three forms: (1) hypercholesterolemia, i.e., an elevatedcholesterol level; (2) hypertriglyceridemia, i.e., an elevatedtriglyceride level;

and (3) combined hyperlipidemia, i.e., a combination ofhypercholesterolemia and hypertriglyceridemia. The term “dyslipidemia”refers to abnormal levels of lipoproteins in blood plasma including bothdepressed and/or elevated levels of lipoproteins (e.g., elevated levelsof LDL, VLDL and depressed levels of HDL).

In one embodiment, the disclosure relates to a method selected fromreducing cholesterol levels or modulating cholesterol metabolism,catabolism, absorption of dietary cholesterol, and reverse cholesteroltransport in a subject, comprising administering to the subject in needthereof an effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof.

In another embodiment, the disclosure relates to a method of treating orpreventing a disease affecting cholesterol, triglyceride, or bile acidlevels in a subject, comprising administering to the subject in needthereof an effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof.

In one embodiment, the disclosure relates to a method of loweringtriglycerides in a subject, comprising administering to the subject inneed thereof an effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof.

In one embodiment, the disclosure relates to a method of preventing adisease state associated with an elevated cholesterol level in asubject. In one embodiment, the disease state is selected from coronaryartery disease, angina pectoris, carotid artery disease, strokes,cerebral arteriosclerosis, and xanthoma.

In one embodiment, the disclosure relates to a method of treating orpreventing a lipid disorder in a subject, comprising administering tothe subject in need thereof an effective amount of a compound of FormulaI or a pharmaceutically acceptable salt thereof.

In one embodiment, the disclosure relates to a method of treating orpreventing one or more symptoms of disease affecting lipid metabolism(i.e., lipodystrophy) in a subject, comprising administering to thesubject in need thereof an effective of a compound of Formula I or apharmaceutically acceptable salt thereof. In one embodiment, thedisclosure relates to a method of treating one or more symptoms of adisease affecting lipid metabolism. In one embodiment, the disclosurerelates to a method of preventing one or more symptoms of a diseaseaffecting lipid metabolism. In one embodiment, the disclosure relates toa method of decreasing lipid accumulation in a subject.

In one embodiment, the disclosure relates to a method of treating,preventing or modulating gastrointestinal disease in a subject,comprising administering to the subject in need thereof an effectiveamount of a compound of Formula I or a pharmaceutically acceptable saltthereof. In some embodiments, the gastrointestinal disease is selectedfrom inflammatory bowel disease (IBD), irritable bowel syndrome (IBS),bacterial overgrowth, malabsorption, post-radiation colitis, andmicroscopic colitis. In one embodiment, the inflammatory bowel diseaseis selected from Crohn's disease and ulcerative colitis.

In one embodiment, the disclosure relates to a method of treating,preventing or modulating renal disease in a subject, comprisingadministering to the subject in need thereof an effective amount of acompound of Formula I or a pharmaceutically acceptable salt thereof. Insome embodiments, the renal disease is selected from diabeticnephropathy, focal segmental glomerulosclerosis (FSGS), hypertensivenephrosclerosis, chronic glomerulonephritis, chronic transplantglomerulopathy, chronic interstitial nephritis, and polycystic kidneydisease.

In one embodiment, the disclosure relates to a method of treating orpreventing metabolic disease in a subject, comprising administering tothe subject in need thereof an effective amount of a compound of FormulaI or a pharmaceutically acceptable salt thereof. In one embodiment, themetabolic disease is selected from insulin resistance, hyperglycemia,diabetes mellitus, diabesity, and obesity. In one embodiment, thediabetes mellitus is type I diabetes. In one embodiment, the diabetesmellitus is type II diabetes.

Diabetes mellitus, commonly called diabetes, refers to a disease orcondition that is generally characterized by metabolic defects inproduction and utilization of glucose which result in the failure tomaintain appropriate blood sugar levels in the body.

In the case of type II diabetes, the disease is characterized by insulinresistance, in which insulin loses its ability to exert its biologicaleffects across a broad range of concentrations. This resistance toinsulin responsiveness results in insufficient insulin activation ofglucose uptake, oxidation and storage in muscle and inadequate insulinrepression of lipolysis in adipose tissue and of glucose production andsecretion in liver. The resulting condition is elevated blood glucose,which is called “hyperglycemia”. Uncontrolled hyperglycemia isassociated with increased and premature mortality due to an increasedrisk for microvascular and macrovascular diseases, including retinopathy(the impairment or loss of vision due to blood vessel damage in theeyes); neuropathy (nerve damage and foot problems due to blood vesseldamage to the nervous system); and nephropathy (kidney disease due toblood vessel damage in the kidneys), hypertension, cerebrovasculardisease, and coronary heart disease. Therefore, control of glucosehomeostasis is a critically important approach for the treatment ofdiabetes.

Insulin resistance has been hypothesized to unify the clustering ofhypertension, glucose intolerance, hyperinsulinemia, increased levels oftriglyceride and decreased HDL cholesterol, and central and overallobesity. The association of insulin resistance with glucose intolerance,an increase in plasma triglyceride and a decrease in high-densitylipoprotein cholesterol concentrations, hypertension, hyperuricemia,smaller denser low-density lipoprotein particles, and higher circulatinglevels of plasminogen activator inhibitor-1, has been referred to as“Syndrome X”. Accordingly, methods of treating or preventing anydisorders related to insulin resistance including the cluster of diseasestates, conditions or disorders that make up “Syndrome X” are provided.

In one embodiment, the invention relates to a method of treating,preventing or ameliorating metabolic syndrome in a subject, comprisingadministering to the subject in need thereof an effective amount of acompound of the disclosure or a pharmaceutically acceptable saltthereof.

In some embodiments, the disclosure relates to a method of treating,preventing, ameliorating or modulating cancer in a subject, comprisingadministering to the subject in need thereof an effective amount of acompound of Formula I or a pharmaceutically acceptable salt thereof. Insome embodiments, the cancer is selected from hepatocellular carcinoma(HCC), also known as malignant hepatoma, cholangiocellular carcinoma,colorectal cancer, gastric cancer, renal cancer, prostate cancer,adrenal cancer, pancreatic cancer, breast cancer, bladder cancer,salivary gland cancer, ovarian cancer, uterine body cancer, and lungcancer.

In one of the embodiments, the hepatocellular carcinoma is selected fromthe group consisting of early stage hepatocellular carcinoma,non-metastatic hepatocellular carcinoma, primary hepatocellularcarcinoma, advanced hepatocellular carcinoma, locally advancedhepatocellular carcinoma, metastatic hepatocellular carcinoma,hepatocellular carcinoma in remission, or recurrent hepatocellularcarcinoma.

In another embodiment, at least one of an agent selected from Sorafenib,Sunitinib, Erlotinib, or Imatinib is co-administered with thecrystalline form of the present disclosure to treat cancer. In oneembodiment, at least one of an agent selected from abarelix, aldeleukin,allopurinol, altretamine, amifostine, anastozole, bevacizumab,capecitabine, carboplatin, cisplatin, docetaxel, doxorubicin, erlotinib,exemestane, 5-fluorouracil, fulvestrant, gemcitabine, goserelin acetate,irinotecan, lapatinib ditosylate, letozole, leucovorin, levamisole,oxaliplatin, paclitaxel, panitumumab, pemetrexed disodium, profimersodium, tamoxifen, topotecan, and trastuzumab is co-administered withthe compound of the invention to treat cancer.

Appropriate treatment for cancers depends on the type of cell from whichthe tumor derived, the stage and severity of the malignancy, and thegenetic abnormality that contributes to the tumor.

Cancer staging systems describe the extent of cancer progression. Ingeneral, the staging systems describe how far the tumor has spread andputs patients with similar prognosis and treatment in the same staginggroup. In general, there are poorer prognoses for tumors that havebecome invasive or metastasized.

In one type of staging system, cases are grouped into four stages,denoted by Roman numerals I to IV. In stage I, cancers are oftenlocalized and are usually curable. Stage II and IIIA cancers are usuallymore advanced and may have invaded the surrounding tissues and spread tolymph nodes. Stage IV cancers include metastatic cancers that havespread to sites outside of lymph nodes.

Another staging system is TNM staging which stands for the categories:Tumor, Nodes, and Metastases. In this system, malignancies are describedaccording to the severity of the individual categories. For example, Tclassifies the extent of a primary tumor from 0 to 4 with 0 representinga malignancy that does not have invasive activity and 4 representing amalignancy that has invaded other organs by extension from the originalsite. N classifies the extent of lymph node involvement with 0representing a malignancy with no lymph node involvement and 4representing a malignancy with extensive lymph node involvement. Mclassifies the extent of metastasis from 0 to 1 with 0 representing amalignancy with no metastases and 1 representing a malignancy withmetastases.

These staging systems or variations of these staging systems or othersuitable staging systems may be used to describe a tumor such ashepatocellular carcinoma. Few options only are available for thetreatment of hepatocellular cancer depending on the stage and featuresof the cancer. Treatments include surgery, treatment with Sorafenib, andtargeted therapies. In general, surgery is the first line of treatmentfor early stage localized hepatocellular cancer. Additional systemictreatments may be used to treat invasive and metastatic tumors.

In one embodiment, the disclosure relates to a method of treating,preventing or ameliorating gallstones in a subject, comprisingadministering to the subject in need thereof an effective amount of acompound of Formula I or a pharmaceutically acceptable salt thereof.

Presence of gallstones in the gallbladder may lead to acutecholecystitis, an inflammatory condition characterized by retention ofbile in the gallbladder and often secondary infection by intestinalmicroorganisms, predominantly Escherichia coli, and Bacteroides species.Presence of gallstones in other parts of the biliary tract can causeobstruction of the bile ducts, which can lead to serious conditions suchas ascending cholangitis or pancreatitis.

In one embodiment, the disclosure relates to a method of treating,preventing or ameliorating a cholesterol gallstone disease.

In one embodiment, the disclosure relates to a method of treating orpreventing neurological disease in a subject, comprising administeringto the subject in need thereof an effective amount of a compound ofFormula I or a pharmaceutically acceptable salt thereof. In oneembodiment, the neurological disease is stroke.

In one embodiment, the disclosure relates to a method of regulating theexpression level of one or more genes involved in bile acid homeostasis.

In one embodiment, the disclosure relates to a method of down regulatingthe expression level of one or more genes selected from CYP7α1 andSREBP-IC in a cell by administering to the cell a crystalline form ofOCA. In one embodiment, the disclosure relates to a method of upregulating the expression level of one or more genes selected from OSTα,OSTβ, BSEP, SHP, UGT2B4, MRP2, FGF-19, PPARγ, PLTP, APOCII, and PEPCK ina cell by administering to the cell a crystalline form of the invention.

The amount of a compound of Formula I or a pharmaceutically acceptablesalt thereof which is required to achieve the desired biological effectwill depend on a number of factors such as the use for which it isintended, the means of administration, and the recipient, and will beultimately at the discretion of the attendant physician or veterinarian.In general, a typical daily dose for the treatment of a FXR mediateddisease and condition, for instance, may be expected to lie in the rangeof from about 0.01 mg/kg to about 100 mg/kg. This dose may beadministered as a single unit dose or as several separate unit doses oras a continuous infusion. Similar dosages would be applicable for thetreatment of other diseases, conditions and therapies including theprevention and treatment of cholestatic liver diseases.

In some embodiment, the disclosure relates to a method of using aneffective amount of a compound of Formula I (including compounds ofFormula II, III, IV, V, VI, VII and VIII) or a pharmaceuticallyacceptable salt thereof as metabolic or pharmacokinetic probe. Incertain embodiments, the disclosure relates to a method of using aneffective amount of a compound of Formula I or a pharmaceuticallyacceptable salt thereof as metabolic probe. In certain embodiments, thedisclosure relates to a method of using an effective amount of acompound of Formula I or a pharmaceutically acceptable salt thereof aspharmacokinetic probe.

The disclosure also relates to the manufacture of a medicament fortreating, preventing, ameliorating or modulating a disease or condition(e.g., a disease or condition mediated by FXR or TGR5), wherein themedicament comprises a compound of Formula I or a pharmaceuticallyacceptable salt thereof.

The disclosure also relates to the manufacture of a medicament fortreating, preventing, ameliorating or modulating a disease or condition(e.g., a disease or condition mediated by FXR or TGR5), wherein themedicament comprises composition comprising a compound of Formula I or apharmaceutically acceptable salt thereof and a pharmaceutally acceptableexcipient.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present application areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentapplication. The examples do not limit the claimed application. Based onthe present disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the presentapplication.

EXAMPLES Example 1: d₅-OCA

Method 1:

3-Keto-OCA (52.5 g, 125.6 mmol), prepared by oxidizing OCA withTPAP/NMO, was dissolved in 400 mL of D₂O and NaOD (40% in D₂O, 35 mL)was added. The mixture was stirred at 90° C. for 4 hours. NaBD₄ (7.77 g,185 mmol) was added portionwise and stirring continued for 2 hours at90° C. The mixture was cooled to room temperature and quenched withaqueous citric acid. The product was extracted with EtOAc (2×). Thecombined organic layers were washed with brine, dried over Na₂SO₄ andconcentrated. The material was taken up in 1 L of water and 10 g ofNaOH. The solution was slowly acidified with 10% HCl until pH 2. Theprecipitate is collected and washed with water and dried in vacuo.

Another batch of 3-keto-OCA (18.4 g, 44 mmol) was converted and combinedwith the first batch. The combined batches had a HPLC-purity around 95%.Pure d₅-OCA was obtained by means of column chromatography (silica;DCM/MeOH 2-10%). The pure material, d₅-OCA, was dissolved in 3 L ofwater containing 16 g of NaOH, and the solution was slowly acidifiedwith 10% HCl (aq) until pH 2. The precipitate was filtered and washedwith water, then dried in vacuo (43 g, 61%). Chemical purity: 99.4%;Isotopic purity: >95%. ¹H NMR (300 MHz, CD₃OD) confirmed the identity ofthis compound (FIG. 1).

Method 2:

Alternatively, deuterium can be incorporate into positions 2 (2d), 4(2d), and 6 (1d) by reducing the diketone using NaBH₄ to generated₅-OCA.

Example 2: d₇-OCA

3,7-diketo-OCA (100 mg, 0.24 mmol) was dissolved in 0.75 mL of D₂O and10 drops of NaOD (40% in D₂O) were added. The mixture was stirred for 2hours at 90° C. then NaBD₄ (21 mg, 0.50 mmol) was added. Stirringcontinued at 90° C. for another 2 hours. The mixture was cooled to roomtemperature and quenched with aqueous citric acid. The product wasextracted with EtOAc (2×). The combined organic layers were washed withbrine, dried over Na₂SO₄ and concentrated. The crude material waspurified by means of column chromatography (silica; chloroform/HOAc9:1). The appropriate fractions were collected and concentratedredissolved in methanol and precipitated with water. After concentrationd₇-OCA was obtained as a white solid (75 mg, 75%). 41 NMR (300 MHz,CD₃OD) confirmed the identity of this compound (FIG. 2).

Example 3: C23(d₂)-OCA

OCA-OMe (methyl ester) prepared from OCA using methanol and HCl orp-toluenesulfonic acid. Sodium (0.4 g, 17.8 mmol) was added to 15 mL ofCH₃OD and dissolved completely before the OCA-OMe (3.5 g, 8.1 mmol) wasadded. The mixture was stirred at 70° C. overnight. The solvent isevaporated and fresh CH₃OD (15 mL) was added. The mixture was refluxedfor another 6 hours and cooled to room temperature. NaOD (40% in D20, 1mL) was added and the mixture was stirred overnight. Afterconcentration, the material was redissolved in chloroform with 2% HOAcand concentrated again. The crude material was purified by means ofcolumn chromatography (silica; DCM/MeOH 5-10%) affording d₂-OCA as awhite solid (2.6 g, 62%). ¹H NMR (300 MHz, CD₃OD) confirmed the identityof this compound (FIG. 3).

Example 4: C3(d)-C7(d)-C23(d₂)-OCA

3,7-Diketo-(d₂)OCA (1.6 g, 3.8 mmol) was dissolved in 10 mL of H₂O andNaOH (0.15 g, 3.8 mmol) was added. The mixture was warmed to 90° C. andNaBD₄ (0.64 g, 15.3 mmol) was added. Stirring continued at 90° C. foranother 2 hours. The mixture was cooled to room temperature and quenchedwith aqueous citric acid. The product was extracted with EtOAc (2×). Thecombined organic layers were washed with brine, dried over Na₂SO₄ andconcentrated. The crude material was purified by means of columnchromatography (silica; DCM/MeOH 5-10%) affording d₄-OCA as a whitesolid (1.1 g, 69%). ¹H NMR (300 MHz, CD₃OD) confirmed the identity ofthis compound (FIG. 4).

Example 5: C2(2d)-C4(2d)-OCA

3-Keto-OCA (5 g, 12.0 mmol), prepared as described in Example 1, wasdissolved in 40 mL of D₂O and NaOD (40% in D₂O, 3.5 mL) was added. Themixture was stirred at 90° C. for 4 hours. NaBH₄ (0.67 g, 17.7 mmol) wasadded portionwise and stirring continued for 2 hours at 90° C. Themixture was cooled to room temperature and quenched with aqueous citricacid. The product was extracted with EtOAc (2×). The combined organiclayers were washed with brine, dried over Na₂SO₄ and concentrated. Thematerial was taken up in 100 mL of water and 1.0 g of NaOH. The solutionwas slowly acidified with 10% HCl (aq) until pH 2. The precipitate wascollected and washed with water and dried in vacuo.

Example 6: Deuterated Glycine Conjugate of (d₅)-OCA, d₅-OCA-O (d₂)Gly

d₅-OCA (2.0 g, 4.7 mmol) was dissolved in 75 mL of DMF and triethylamine(4.7 g, 47 mmol) was added and the mixture was stirred for 5 min.4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.9g, 14.1 mmol) was added and the mixture was stirred for 5 min.d₂-Gly-OMe-HCl (1.77 g, 14.1 mmol) was added and stirring continuedovernight. The mixture was poured into water and extracted with EtOAc(2×). The combined organic layers were washed with brine, dried overNa₂SO₄ and concentrated. The crude material was purified by means ofcolumn chromatography (silica; DCM/MeOH 3-7%) affordingd₅-OCA-O(d₂)Gly-OMe as a white foam (1.6 g, 68%).

d₅-OCA-O (d₂)Gly-OMe (1.6 g, 3.2 mmol) was dissolved in 20 mL of CH₃ODand NaOD (40% in D₂O, 1 mL) was added. The mixture was stirred for 1hour and quenched with citric acid (5 g in 100 mL of water). The productwas extracted with EtOAc (2×). The combined organic layers were washedwith brine, dried over Na₂SO₄ and concentrated. The material was takenup in water with 2 eqv of NaOH. The solution was acidified to pH 1 with1N HCl and the precipitate was collected and dried in vacuo affordingd5-OCA-O(d2)Gly as white solid (1.0 g, 64%). ¹H NMR (300 MHz, CD₃OD)confirmed the identity of this compound (FIG. 5).

Example 7: Deuterated Taurine Conjugate of (d₅)-OCA, d₅-OCA-O (d₄)Tau

d₅-OCA (1.65 g, 3.87 mmol), d₄-Taurine (0.50 g, 3.87 mmol), DIPEA (1.0g, 7.74 mmol) and EEDQ (0.96 g, 3.87 mmol) were dissolved in 25 mL ofDMF and stirred at 90° C. for 2.5 hours. The mixture was cooled to roomtemperature and 5 mL of NaOH was added. The mixture was concentrated andstripped with toluene twice. The residue was dissolved in water (40 mL)and washed with ethyl acetate (3×). The aqueous layer was acidified with6N HCl to pH 1 and washed with diethyl ether (3×) and ethyl acetate(3×). The aqueous layer was partially concentrated till some productoils out. The mixture was allowed to stand for a while and the water wasdecanted from the oily product. The oily product was concentrated andstripped with CH₃CN/MeOH twice. The off-white solid was purified byRP-ISCO (RP=reversed phase). The appropriate fractions were collected,concentrated and stripped with CH₃CN/MeOH twice affording d₅-OCA-O(d₄)Tau as white solid (1.3 g, 62%). 41 NMR (300 MHz, CD₃OD) confirmedthe identity of this compound (FIG. 6).

Example 8: OCA-3-O-Glucuronide

Step 1: OCA methyl ester

A solution of OCA (212.7 mg, 0.51 mmol), anhydrous methanol (6 mL), andpara-toluenesulfonic acid (19.6 mg, 0.11 mmol) was sonicated for 2 hoursat which time the reaction was complete by TLC. The solvent wasevaporated via gentle nitrogen and the residue was dried in vacuuo (atroom temperature) for 30 minutes. Chloroform (8 mL) and saturatedaqueous sodium bicarbonate (5.2 mL) were charged and the aqueous layerwas separated. The organic layer was sequentially washed with water (5mL) and brine (5 mL). The organic layer was dried over sodium sulfateand filtered. The filtrate was concentrated via rotary evaporation toafford OCA-methyl ester (227 mg, 100% yield) as a clear oil. ¹H NMRconfirmed the identity of this compound.

Step 2: Synthesis of Intermediate 1

A solution of OCA-methyl ester (227 mg, 0.52 mmol) in anhydrous toluene(8 mL) was treated with silver carbonate (672.7 mg, 2.4 mmol) andacetobromo-α-D-glucuronic acid methyl ester (1034.9 mg, 2.6 mmol). Theresulting solution was stirred at room temperature for four days atwhich time the reaction was complete by HPLC. The reaction mixture wasfiltered through a 1.0 μm PTFE syringe filter and the syringe filter waswashed with ethyl acetate (2×5 mL). The pooled filtrates were evaporatedvia rotary evaporation and the residue was purified via silica gel flashcolumn chromatography to afford crude Intermediate 1 (183 mg). Thestructure of Intermediate 1 was confirmed by ¹H NMR, ¹³C NMR and HMBCNMR. Glucuronide is in 3 position as demonstrated by HMBC couplingbetween C3 proton (3.45 ppm) and the 1′ anomeric carbon (99 ppm).

Step 3: Synthesis of OCA-3-O-Glucuronide

A solution of intermediate 1 (150 mg, 0.2 mmol) in methanol (12 mL) wastreated with 6.5 mL of aqueous 2M LiOH (13 mmol). The resulting solutionwas stirred for 2 hours at room temperature at which time the reactionwas complete by TLC. The solvents were evaporated via slow nitrogenstream. Water (2 mL) was added and concentrated hydrochloric acid wasadded dropwise until the pH of the solution was 4-5 (as measured bynarrow range pH paper). The solvents were evaporated via rotaryevaporation and the resulting residue was dried in vacuuo overnight. Aslurry of crude product in 3 mL of 0.1% (v/v %, acetic acid in water)was applied to a pre-washed (washed with 3 column volumes of methanolfollowed by 5 column volumes of 0.1% acetic acid in water) 10 gram C18Sep-Pak® cartridge. The pooled product-containing fractions (asdetermined by TLC) were evaporated via rotary evaporation and dried invacuuo to afford OCA-3-O-Glucuronide (64 mg, 0.1 mmol, 50% yield fromIntermediate 1, 10% overall yield from OCA). OCA-3-O-Glucuronide wasanalized by ¹ H NMR (Table 1 and FIG. 7).

TABLE 1 ¹H NMR of OCA-3-O-Glucuronide 600 MHz NMR Metabolite Chemicalgroups Chemical Shift ppm Solvent, MHz C-18 H₃ s, 0.69INT-747-3-glucuronide C-19 H₃, C-26 H₃ m, 0.89 CH₃OD, 600 C-21 H₃ d,0.95 C-23 H₁ m, 2.19 C-23 H₁ m, 2.34 C-3 H₁ m, 3.18 C-2′, C-3′, C-4′, H₃m, 3.51; t, 3.37 C-7 H₁ t, 3.65 C-5′ H₁ d, 3.73 C-1′ H1 d, 4.43 (J_(H-H)= 7.8 Hz)

Example 9: [³H]OCA-24-Glucuronide

Step 1: Synthesis of [³H]OCA

A solution of 7-ketone (217.6 mg, 0.52 mmol) in water (2.4 mL) and 50%aqueous NaOH (0.21 mL) was heated to 90° C. to afford solution A. Theresulting solution was transferred into a dry reaction tube containingNaBT₄ (7.4 mg, 0.19 mmol, 49 mCi). The tube was sealed and heated at 90°C. for 1 hour. Additional unlabeled NaBH₄ (75 mg, 2.0 mmol) was addedand the heating was continued at 90° C. for 3 additional hours. Theresulting mixture was cooled to room temperature and diluted with amixture of n-butyl-acetate (3 mL) and a solution of citric acid (1.329g, 6.92 mmol) in water (1. 7 mL). The layers were separated and theaqueous phase was discarded. The reaction was repeated using 240.6 mg7-ketone and 5.7 mg of NaBT₄.

The pooled organic layers from the two reactions were evaporated andpurified via silica gel flash column chromatography eluting with 1/2(v/v, hexanes/ethyl acetate) to afford [³H]-OCA (198 mg, 0.47 mmol, 4.8mCi, 6% yield from NaBT₄, 43% yield from 7-ketone). An HPLC/UVchromatogram of OCA standard and a radio-HPLC chromatogram of [³H]-OCAwere performed.

Step 2: Synthesis of Intermediate 2

A solution of [³H]-OCA (198 mg, 0.47 mmol, 4.8 mCi) and unlabeled OCA(200 mg, 0.48 mmol) in anhydrous DMF (40 mL) was treated with silvercarbonate (754.4 mg, 2.7 mmol) and acetobromo-α-D-glucuronic acid methylester (485.4 mg, 1.2 mmol). The resulting solution was stirred at roomtemperature for four days at which time the composition was 21% product,71% [³H]-OCA, and 8% side product (by radio-HPLC). The reaction mixturewas filtered through a 0.45 μm PTFE syringe filter and the syringefilter was washed with ethyl acetate (2×10 mL). The pooled filtrateswere evaporated via rotary evaporation and the residue was purified viasilica gel flash column chromatography. The pooled product-containingfractions were evaporated via rotary evaporation to afford crudeintermediate 2 (168 mg, 0.23 mmol, 0.87 mCi@ 80% purity with 20%[³H]-OCA by radio-HPLC) which was used in the next step without furtherpurification. The structure of non-labeled intermediate 2 was confirmedby ¹H NMR, ¹³C NMR and HMBC NMR.

Step 3: Synthesis of Intermediate 3

A solution of Intermediate 2 (168 mg) in DMSO (23 mL) and 93 mL of 25 mMcitric acid buffer (pH=5) was treated with LAS enzyme (Supplier=Aldrich,1.245 g) and CSR enzyme (Supplier=Wako chemical, 1.209 g). The resultingsolution was heated at 40° C. for 2 hours at which time the reacton wascomplete by radio-HPLC. The resulting mixture was cooled to roomtemperature and applied to a pre-washed (washed with 3 column volumes ofmethanol followed by 5 column volumes of 0.01% acetic acid in water) 10gram C18 Sep-Pak cartridge. The cartridge was sequentially washed with(3 column volumes each) of 0.01% formic acid in water, 80/20, 50/50,40/60, 30/70, 20/80, and 10/90 (v/v %, 0.01% formic acid inwater/acetonitrile) followed by neat acetonitrile. The pooledproduct-containing fractions (as determined by radio-HPLC) wereevaporated via rotary evaporation and dried in vacuuo to affordintermediate 3 (72.3 mg, 0.12 mmol).

Step 4: Synthesis of [³H]-OCA-24-Glucuronide

A solution of intermediate 3 (72.3 mg, 0.12 mmol) in DMSO (6.4 mL) and70 mL of 25 mM citric acid buffer (pH=5) was treated with Cal-B enzyme(Supplier Aldrich, 42.3 mg). The resulting solution was heated at 40° C.for 3 hours at which time the reaction was complete by radio-HPLC. Theresulting mixture was cooled to room temperature and applied to apre-washed (washed with 3×column volumes of methanol followed by 5column volumes of 0.01% acetic acid in water) 10 gram C18 Sep-Pakcartridge. The cartridge was sequentially washed with (3 column volumeseach) of 0.01% formic acid in water, 90/10, 80/20, 70/30, 60/40, 50/50,and 40/60 (v/v %, 0.01% formic acid in water/acetonitrile). The pooledproduct-containing fractions (as determined by radio-HPLC) wereevaporated via rotary evaporation and dried in vacuuo to afford[³H]-OCA-24-Glucuronide (39 mg, 0.065 mmol, 14% overall yield from[³H]-OCA. Structure of [³H]-OCA-24-Glucuronide was confirmed by ¹H NMR(Table 2 and FIG. 8) and HMBC NMR. Comparison plot for ¹H NMR spectra ofOCA-24-Glucuronide methyl ester and [³H]OCA-24-Glucuronide (expansion of3.0-5.0 ppm region) (FIG. 9) shows that the methyl ester protons(singlet at 3.78 ppm in OCA-24-Glucuronide methyl ester) are not presentin [³H]OCA-24-Glucuronide.

TABLE 2 ¹H NMR of [³H]-OCA-24-Glucuronide 600 MHz NMR MetaboliteChemical groups Chemical Shift ppm Solvent, MHz C-18 H₃ s, 0.69[³H]-INT-747-24- C-19 H₃, C-26 m, 0.91 Glucuronide H₃ C-21 H₃ d, 0.95CH₃OD, 600 C-23 H₁ m, 2.35 C-23 H₁ m, 2.45 C-3 H₁ s, 3.19 C-2′, C-3′,C-4′, H₃ m, 3.46 C-7 H₁ m, 3.65 C-5′ H1 d, 3.79 C-1′ H₁ d, 5.49 (J_(H-H)= 7.8 Hz)

Example 10. [¹⁴C-24]OCA

3,7-Di-TFA-OCA (5.6 g, 9.1 mmol) is dissolved in carbon tetrachloride(112 mL) and stirred in the presence of Pb(OAc)₄ and iodine at 70-75° C.and in the presence of light. Upon reaction completion, the mixture iscooled, washed with aqueous sodium bisulfate, dried over sodium sulfateand concentrated to a residue. The residue was chromatographed on silicagel to generate the 3,7-di-TFA-iodide intermediate (3.2 g, 4.61 mmol).The TFA protecting groups were removed by dissolving the intermediate inmethanol and treating with aqueous potassium carbonate, thus generatingthe diol intermediate (1.85 g, 3.68 mmol) after extractive work up indichloromethane. The diol intermediate (1.3 g, 2.59 mmol) was dissolvedin DMF and treated with potassium [¹⁴C] cyanide (1.1 equiv, 2.85 mmol).Upon reaction completion, aqueous workup and extraction withdichloromethane was performed to generate the nitrile intermediate (1.0g, 2.49 mmol). The nitrile intermediate was refluxed in 10 wt % sodiumhydroxide in ethanol. Upon reaction completion, the mixture wasconcentrated and diluted with water. [¹⁴C-24] OCA was isolated byadjusting the pH with citric acid, extracting with ethyl acetate andconcentrating to a solid (1.0 g, 2.38 mmol).

Example 11. [¹⁴C-24]100

3,7,11-Tri-TFA-100 (5.0 g, 6.9 mmol) is dissolved in carbontetrachloride (100 mL) and stirred in the presence of Pb(OAc)₄ andiodine at 70-75° C. and in the presence of light. Upon reactioncompletion, the mixture is cooled, washed with aqueous sodium bisulfite,dried over sodium sulfate and concentrated to a residue. The residue waschromatographed on silica gel to generate the 3,7,11-tri-TFA-iodideintermediate (2.8 g, 3.45 mmol). The TFA protecting groups were removedby dissolving the intermediate in methanol and treating with aqueouspotassium carbonate, thus generating the triol intermediate (1.45 g,2.79 mmol) after extractive work up in dichloromethane. The triolintermediate was dissolved in DMF and treated with potassium [¹⁴C]cyanide (1.1 equiv, 3.07 mmol). Upon reaction completion, aqueous workupand extraction with dichloromethane was performed to generate thenitrile intermediate (1.0 g, 2.48 mmol). The nitrile intermediate wasrefluxed in 10 wt % sodium hydroxide in ethanol. Upon reactioncompletion, the mixture was concentrated and diluted with water.[¹⁴C-24] 100 was isolated by adjusting the pH with citric acid,extracting with ethyl acetate and concentrating to a solid (1.0 g, 2.33mmol).

Example 12: [¹⁴C]OCA at High Specific Activity (Synthesis was CarriedOut on a 105 mCi Scale)

Step 1: Preparation of compound C2

Diisopropylamine and tetrahydrofuran (THF) are stirred at approximately−20° C. under argon. N-butyllithium is added slowly and the mixturestirred at approximately −20° C. before cooling to approximately −70° C.Chlorotrimethylsilane is added and the mixture stirred. Compound C1dissolved in THF is added slowly and stirred at approximately −70° C.and then warmed to approximately −50° C. and further stirred. Aqueoussodium hydrogen carbonate is added and the mixture warmed to roomtemperature. The aqueous layer is separated and washed with ethylacetate. The organic layers are combined and washed with aqueous sodiumhydrogen carbonate, water and brine and dried over sodium sulphate. Thesolution is filtered, washed with toluene, rotary evaporated anddesiccated under vacuum to yield compound C2 as a yellow oil.

Step 2: Preparation of Compound [¹⁴C]C3

[1-¹⁴C] Acetaldehyde, compound C2 and dichloromethane are stirred atapproximately-70° C. under nitrogen. Boron trifluoride etherate is addedslowly and the reaction stirred at approximately −70° C. and then atroom temperature overnight. Nitrogen is bubbled through the solution.The solution is then cooled in an ice bath and aqueous sodium hydrogencarbonate is added. The aqueous layer is separated and washed withdichloromethane. The organic layers are combined and dried over sodiumsulphate. The solution is filtered, rotary evaporated and the residue ispurified by flash chromatography (silica, hexane:ethyl acetate). Thesolution is rotary evaporated, methanol added and rotary evaporated toyield [¹⁴C] C3 as a gum.

Step 3: Preparation of Compound [¹⁴C]C4

Compound [′C]C3, methanol and sodium hydroxide are stirred atapproximately 45° C. The reaction is cooled, partially rotary evaporatedto remove methanol, and water is added. The solution is cooled in an icebath and acidified with aqueous phosphoric acid. The mixture isextracted with dichloromethane. The organic layers are combined anddried over sodium sulphate. The solution is filtered, rotary evaporatedand desiccated under vacuum to yield compound [¹⁴C]C4 as a solid.

Step 4: Preparation [¹⁴C]OCA Ketone C5

[¹⁴C]C4 is dissolved in sodium hydroxide, 5% palladium on carbon isadded, and hydrogenated at room temperature and then at approximately115° C. The reaction is cooled to room temperature, filtered and cooledin ice. The solution is acidified with concentrated hydrochloric acidand extracted with dichloromethane. The dichloromethane solution isdried over sodium sulphate, filtered and rotary evaporated. The residueis dissolved in ethanol:acetonitrile:water to give a solution containing[¹⁴C]OCA ketone C5. This solution was purified by HPLC (C18 column,aqueous hydrochloric acid:acetonitrile). The solution is rotaryevaporated, water is added and the solution is rotary evaporated again.The solid is desiccated under vacuum to yield [¹⁴C]OCA ketone C5.

The quality of the radiolabelled precursor [¹⁴C]OCA ketone C5 isconsidered to be critical to the quality of [¹⁴C]OCA C6 drug substanceand is therefore controlled according to the results given in Table 3.

TABLE 3 Critical intermediate for synthesis of [¹⁴C]OCA C6 ComponentSupplier Analysis Results Radiolabelled Quotient Specific activity 25mCi mmol⁻¹ designated starting Bioresearch Purity by HPLC UV 99.5%material (Radiochemicals) (UV and RCP) RCP 99.3 % [¹⁴C]OCA Ketone LtdCo-chromatography with Co-chromatographs reference (HPLC) Conforms NMRspectrometry Conforms Mass spectrometry

Step 5: Preparation of [¹⁴C]OCA C6

[¹⁴C]OCA ketone C5 is suspended in 2M sodium hydroxide and heated toapproximately 80° C. A solution of sodium borohydride in water is addedand the resulting reaction mixture is heated at approximately 100° C.The progress of the reaction is monitored by TLC. Water anddichloromethane are added to the reaction mixture. To quench thereaction ortho-phosphoric acid is slowly added whilst stirring until noeffervescence occurs and no solid remains. The aqueous layer isseparated and extracted with dichloromethane. The organic layers arecombined, dried over sodium sulphate, filtered and evaporated to yieldcrude [¹⁴C]OCA C6 as a white solid. Crude [¹⁴C]OCA C6 is dissolved inthe minimum volume of ethyl acetate and diluted with an equal volume ofheptane. The solution is loaded onto a silica column and eluted withheptane: ethyl acetate (1% acetic acid in ethyl acetate). The fractionsare collected and analysed by TLC. Fractions containing [¹⁴C]OCA C6 arecombined, washed with water, dried with sodium sulphate and concentratedunder reduced pressure to remove solvent.

The material from the flash column is dissolved in dichloromethane andstirred for at least 16 hours with QuadraPure Tu palladium scavenger.The scavenger is removed by filtration and filtrate concentrated underreduced pressure to remove solvent. The material from the filtrate isfurther purified using preparative HPLC with a C18 column eluting with 5mM sodium phosphate:acetonitrile:methanol. The fractions containing[¹⁴C]OCA C6 are combined and the organic solvent is removed underreduced pressure. The remaining aqueous solution is extracted with ethylacetate. The ethyl acetate extracts are combined, washed with brine andthe solvent is removed portion-wise under reduced pressure.

Purified [¹⁴C]OCA C6 is dissolved in 0.2 M sodium hydroxide understirring. The solution is filtered, washed with water and stored at roomtemperature. Separately 0.2 M hydrochloric acid is stirred and filtered.The solution of [¹⁴C]OCA C6 in sodium hydroxide is added to thehydrochloric acid drop wise. The resulting suspension is stirred and thesolid collected by centrifugation. The solid is washed with water,centrifuged and the solid is then transferred to a sinter funnel anddried under suction. The resulting dried product is [¹⁴C]OCA C6 at highspecific activity.

[¹⁴C]OCA was characterised by mass spectrometry, HPLC and ¹H NMR. Thedata show that the end product of the technical synthesis, [¹⁴C]OCA drugsubstance was 99.6% chemically pure and 98.0% radiochemically pure,which is considered to be of suitable quality for clinical use. Thebatch also met the specific activity requirements (result obtained 299.4kBq mg-1).

The ¹H NMR spectrum obtained from [¹⁴C]OCA (FIG. 10) was compared withthe ¹H NMR spectrum obtained from OCA Reference Material (FIG. 11).

Mass Spectrum for [¹⁴C]OCA (MeOH:0.5% ammonia, 9:1, 10 μg/mL, 20 μL/mininf,

ES-, cone=85 V, Temp=80° C.):Chemical formula (unlabelled): C26H₄₄O₄

Exact Mass: 420.32

m/z Intensity Assignment 419.3 1.26 × 10⁸ [M—H]⁻ unlabelled 420.3 3.67 ×10⁷ [M—H]⁻ 1 × C-13 421.3 1.35 × 10⁷ [M—H]⁻ 1 × C-14 label or 2 × C-13451.5 1.41 × 10⁷ [(M—H)MeOH]⁻ unlabelled 452.4 4.24 × 10⁶ [(M—H)MeOH]⁻ 1× C-13 453.4 1.57 × 10⁶ [(M—H)MeOH]⁻ 1 × C-14 label or 2 × C-13

Mass Spectrum for Reference Standard OCA (MeOH:0.5% ammonia, 9:1, 10μg/mL, 20 μL/min inf, ES-, cone=85 V, Temp=80° C.):

m/z Intensity Assignment 419.3 1.30 × 10⁸ [M—H]⁻ 420.3 5.12 × 10⁷ [M—H]⁻1 × C-13 421.3 8.54 × 10⁶ [M—H]⁻ 2 × C-13 451.4 1.86 × 10⁷ [(M—H)MeOH]⁻1 × C-13 452.4 5.59 × 10⁶ [(M—H)MeOH]⁻ 2 × C-13

Example 13: Clinical Study

The objectives of this study were to determine the absolutebioavailability of OCA, and evaluate the absorption, distribution,metabolism, and excretion of OCA in healthy male subjects. In addition,the oral pharmacokinetics (PK) of OCA and carbon-14 ([¹⁴C]-OCA, andintravenous (IV) PK of [¹⁴C]-OCA were evaluated.

The Simplified Investigational Medicinal Product Dossier (sIMPD)describes the [¹⁴C]-OCA Solution for Intravenous Administration (20μg/mL), the [¹⁴C]-OCA Drug in Capsule and the OCA Tablet. Thenon-radiolabelled OCA drug substance and drug product (OCA Tablet (25mg)) were used in this study.

In order to obtain [¹⁴C]OCA at the specific activity required forclinical use, [¹⁴C]OCA is synthesised at high specific activity and isthen radiodiluted using non-radiolabelled OCA to give the final [¹⁴C]OCAdrug substance at the correct specific activity for use in the proposedclinical trial. The described above synthesis was carried out on a 105mCi scale. [¹⁴C]OCA high specific activity component can be combinedwith the OCA cold component at an approximate ratio of 1:6, [¹⁴C]OCA toOCA to produce [¹⁴C]OCA drug substance with the specific activityrequired for use in the [¹⁴C]-OCA Solution for intravenousadministration (e.g., 20 μg/mL) or oral administration in clinical trialor other study. The material can then further diluted by a factor of 1:2(overall 1:12) [¹⁴C]OCA to OCA to produce [¹⁴C]OCA drug substance withthe specific activity required for use in the [¹⁴C]-OCA Drug in Capsulein clinical trial or other study.

Absolute Bioavailability of OCA

Absolute bioavailability is the percentage of the administered dose thatis absorbed into the systemic circulation unchanged. Data in humansubjects to date indicate that the PK profile of OCA is consistent withthat expected of a bile acid. There is rapid absorption, followed byextensive conjugation (metabolism) to glycine and taurine to formglyco-OCA and tauro-OCA, respectively, which undergo extensiveenterohepatic circulation. Plasma concentrations dramatically increaseshortly after food intake, consistent with gallbladder emptying into theduodenum. A small percentage the conjugates are subsequentlydeconjugated to re-form the parent drug (OCA) in the colon. Notably,glyco-OCA and tauro-OCA have been shown to have equipotentpharmacological action to that of the parent drug, thus, their plasma PKprofiles are of interest. The total metabolite profile was alsoinvestigated in this study.

(a) IV PK as part of Absolute Bioavailability Assessment

The PK objectives of Part 1 were to determine the absolutebioavailability of OCA, to define the intravenous (IV) PK of amicrotracer dose of [14C] OCA, and to evaluate the oral PK of OCA. InPart 1, subjects were administered the oral dose (Regimen A: 25 mg OCAtablet), followed by the IV microtracer dose (Regimen B: 100 μg[14C]-OCA) 1 hour 45 minutes after the oral dose administration. The 15minute IV infusion ended at the estimated tmax for the oral dose (2hours).

Mean plasma concentrations of total radioactivity are shown in FIG. 12,and of [¹⁴C]-OCA in FIG. 13. The highest concentration of radioactivityin plasma was measured after completion of the IV dose, and theconcentrations of OCA declined rapidly within 4 hours. After the initialdecline, a low-level, prolonged profile of OCA and total radioactivitywas observed, as expected due to enterohepatic recirculation.Concentrations of total radioactivity demonstrated exposure peaks inmeasurements taken at 36 and 60 hours, likely due to the conjugatesbeing released from the gallbladder following meals.

Pharmacokinetic parameters of [¹⁴C]-OCA and total radioactivity aresummarized in Table 4. The AUC_(0-t) of [¹⁴C]OCA accounted forapproximately 20% of the total radioactive exposure over 72 hours,indicating that the formation of OCA conjugates (glyco-OCA, tauro-OCA,and potentially other metabolites) accounted for the majority of thecirculating radioactivity. Also, [¹⁴C]-OCA showed a moderate clearancevalue and a relatively low volume of distribution.

TABLE 4 Mean (SD) of IV Plasma Pharmacokinetic Parameters: Regimen B,Study Part 1: PK Population (N = 5) OCA Part 1 IV [14C]-OCA TotalRadioactivity Parameter N = 5 N = 5 C_(max) (ng/mL) 9.71 (0.3279) 9.13(0.5512) AUC_(0-t) (hours * ng/mL) 3.86 (0.1738) 18.5 (4.234)  CL(L/hours) 25.0 (1.052)  N/A V_(z) (L) 618 (341.9)  N/A V_(ss) (L) 210(62.14)  N/A AUC_(0-t) = Area under the concentration versus time curvefrom time zero to the last sampling time with quantifiable analyte; CL =total plasma clearance; C_(max) = Maximum observed analyte concentrationin plasma; IV = Intravenous; N/A = Not applicable; OCA = Obeticholicacid; V_(ss) = Volume of distribution at steady state; V_(z) = Volume ofdistribution; Regimen B = 15 minute IV infusion of 100 μg [¹⁴C]-OCA

(b) Oral PK As Part of Absolute Bioavailability Assessment

To evaluate the oral PK, plasma concentrations of OCA and its conjugateswere measured following an oral dose (Regimen A: 25 mg OCA). Mean (SD)plasma concentrations over time of OCA are presented in FIG. 14 and PKparameters are presented in Table 5. OCA was rapidly absorbed; themedian tmax was 0.5 hrs. Following Cmax, OCA concentration declinedrapidly within 4 hours, consistent with the IV profile of OCA.Subsequent OCA concentrations remained, on average, less than 10% ofCmax up to 72 hours post-dose. A prolonged profile of OCA was observedover the course of 72 hours (FIG. 4). This low level persistence ofparent OCA is most likely due to deconjugation of glyco-OCA andtauro-OCA by commensal bacteria in the ileum and colon, followed byreabsorption of OCA.

Mean (SD) plasma concentrations over time of total OCA (the sum of OCA,glyco-OCA, and tauro-OCA) are presented in FIG. 15 (Note: Total OCAcalculated as sum of OCA, glyco-OCA, and tauro-OCA concentrations ateach time point, glyco-OCA, and tauro-OCA concentrations at each timepoint expressed as mass equivalents of OCA). Pharmacokinetic parametersof OCA, glyco-OCA, tauro-OCA, and total OCA are summarized in Table 5.Consistent with the [¹⁴C]-OCA results, the maximum exposure of total OCAoccurred after a meal, consistent with gallbladder release andenterohepatic recirculation, leading to exposure levels being observedup to 72 hours post-dose. Based on overall exposure of total OCA(AUC_(0-t)), glyco-OCA and tauro-OCA are the primary components ofexposure, with minimal contribution (less than 10%) from the parent(OCA).

TABLE 5 Mean (SD) Oral Plasma Pharmacokinetic Parameters (Regimen A,Study Part 1: PK Population (N = 5)) Parameter OCA Glyco-OCA Tauro-OCATotal OCA^(a) t_(max) (h)^(b) 0.500 8.000 36.000 1.500 (0.50, 2.03)(2.00, 36.00) (2.00, 60.00) (0.50, 11.00) C_(max) (ng/mL) 80.9 (26.41) 57.4 (24.43) 23.1 (7.412)  105 (28.93) AUC_(0-t)  144 (21.06) 1540(480.7)  624 (159.6) 1990 (525.6) (hours * ng/mL) F (%) 17.1 (2.993)AUC_(0-t) = Area under the concentration versus time curve from timezero to the last sampling time with quantifiable analyte; C_(max) =Maximum observed analyte concentration in plasma; F = Absolutebioavailability as determined by the ratio of dose normalized AUC fororal dose/dose normalized AUC for IV dose; glyco-OCA = glycine conjugateof obeticholic acid; OCA = obeticholic acid; tauro-OCA = taurineconjugate of obeticholic acid; t_(max) = Time of maximum plasmaconcentration ^(a)Total OCA calculated as sum of OCA, glyco-OCA, andtauro-OCA concentrations at each time point expressed as massequivalents of OCA. ^(b)Median and range. Note: Regimen A was a singleoral dose of a 25 mg OCA tablet administered with 240 mL water.

Absolute bioavailability was determined by the ratio of dose normalizedAUC for oral dose/dose normalized AUC for IV dose. As shown in Table 5,the mean absolute bioavailability (F) of OCA was approximately 17%. Thisrelatively low value for bioavailability is consistent with theefficient uptake typical of bile acids into the liver, the primary siteof action of OCA. PK results were as expected for a bile acid. OCA israpidly absorbed in the intestine and taken up in the liver, where it isconjugated with glycine and taurine. Glyco-OCA and tauro-OCA were theprimary components of exposure, with minimal contribution from theparent (OCA). Enterohepatic recirculation produced an extended profile,with maximum exposure occurring after meals.

The results from Part 1 (Absolutely Bioavailability) showed that theoral bioavailability of OCA is 17%. Following an oral dose of 25 mg OCAand an IV microtracer dose of 100 μg [¹⁴C]-OCA, the concentrations oftotal radioactivity and of [¹⁴C]-OCA declined rapidly within the first 4hours, as the concentrations of the conjugates (glyco-OCA, andtauro-OCA) increased and persisted over 72 hours. Exposure of total OCA(sum of OCA, glyco-OCA, and tauro-OCA) peaked after meals, consistentwith gallbladder release and enterohepatic recirculation. Takentogether, these data indicate that OCA is rapidly absorbed in theintestine and taken up in the liver, where it is conjugated with glycineand taurine prior to excretion via the bile. Mass Balance and MetaboliteIdentification (Absorption, distribution, metabolism, and excretion ofOCA (in healthy male subjects)

Preclinical mass balance and radiolabeled single dose PK studies in therat have examined biliary, urinary, and fecal excretion of OCA and itsmetabolites. These results revealed that the radioactivity derived from[¹⁴C] OCA after a single oral administration was excreted mainly in thefeces (due primarily to extensive biliary excretion), and excretion ofOCA appears to be consistent with that of natural bile acids. This studysought to confirm the routes of elimination in humans.

The PK objectives of Part 2 were to assess the mass balance recoveryfrom excreta after an oral dose of 25 mg [¹] OCA, and to assess themetabolite profile of [¹⁴C]-OCA in plasma, urine, and fecal samples.Part 2 also evaluated the extent of distribution of total radioactivityinto blood cells, and identified metabolites accounting for more than10% of circulating total radioactivity.

(a) Oral Pharmacokinetics

The concentrations of total radioactivity and total OCA in plasma overtime are shown in FIG. 16 (Note: Total OCA calculated as sum of OCA,glyco-OCA, and tauro-OCA concentrations at each time point, glyco-OCA,and tauro-OCA concentrations at each time point expressed as massequivalents of OCA) and FIG. 17 (Note: Total OCA calculated as sum ofOCA, glyco-OCA, and tauro-OCA concentrations at each time point,glyco-OCA, and tauro-OCA concentrations at each time point expressed asmass equivalents of OCA. Units for total radioactivity are massequivalent to OCA (i.e. ng-eq OCA/mL)). These concentrations differed bya consistent amount, which suggested the presence of circulating OCAmetabolites other than glyco-OCA and tauro-OCA. Two additional OCAmetabolites were identified: OCA-3 glucuronide and OCA-24 glucuronide,based on the metabolite profiling of radioactivity, mass spectrometry,and authentic standards for the glucuronide metabolites.

Pharmacokinetic parameters of OCA and its conjugates, and totalradioactivity, are summarized in Table 6. Similar to the resultsobserved in Part 1, OCA was rapidly absorbed; the median tmax was 1.25hrs. Following Cmax, OCA concentration declined rapidly within the first4 hours. The maximum exposure of total OCA, including the conjugates,occurred after meals, consistent with gallbladder release andenterohepatic recirculation, leading to exposure levels being observedover the approximately 4-week sampling period.

TABLE 6 Mean (SD) of Plasma Pharmacokinetic Parameters: Regimen C, StudyPart 2: PK Population, (N = 8) OCA Part 2 (N = 8) Total RadioactivityParameter OCA Glyco-OCA Tauro-OCA Total OCA^(a) in Plasma t_(max)(h)^(b) 1.25 10.0 9.03 9.03 1.25 (0.750, 2.50) (3.00, 36.0) (2.50, 36.0)(0.750, 36.0) (0.750, 3.00) C_(max) (ng/mL) 63.7 (19.07)  73.8 (39.96) 24.4 (14.21)  97.8 (39.47)   172 (45.86) AUC_(0-t)  475 (215.5) 4690(2232) 2550 (2155) 6600 (3646) 12700 (5151) (hours * ng/mL) AUC_(0-t) =Area under the concentration versus time curve from time zero to thelast sampling time with quantifiable analyte; C_(max) = Maximum observedanalyte concentration in plasma; glyco-OCA = glycine conjugate ofobeticholic acid; OCA = obeticholic acid; tauro-OCA = taurine conjugateof obeticholic acid; t_(max) = Time of maximum plasma concentrationNote: Regimen C = a 25 mg Oral Dose of [14C]-OCA ^(a)Total OCAcalculated as sum of OCA, glyco-OCA, and tauro-OCA concentrations ateach time point expressed as mass equivalents of OCA. ^(b)Median andrange.

(b) Mass Balance

The mean cumulative recovery (cumulative % Ae (Amount excreted)) ofradioactivity in the urine and feces of subjects receiving oral[¹⁴C]-OCA is shown in FIG. 18. Following a single oral dose of 25 mg[¹⁴C]-OCA a mean of 75.1% (range between 28.3% and 97.5%) of the totalradioactivity administered was recovered from urine and feces by the endof the inpatient sampling period (504 hours postdose). An average of2.83% (range 1.57% to 4.00%) of the total radioactivity was recoveredfrom the urine, and the majority of drug-related material in the urinewas recovered within the first 312 hours after investigational productadministration. An average of 72.3% (range 25.2% to 95.9%) was recoveredfrom feces by 504 hours postdose. However, because only 1 subject hadachieved a cumulative recovery of greater than 90% at 504 hours, theother 7 subjects conducted additional home fecal collections beyond 504hours postdose (⅞ subjects until 816 hours, 3/8 subjects until 888 hourspostdose and 2/8 subjects until 1152 hours postdose). Total recovery(urine and feces combined, sampled up to 1152 hours) from each of thesubjects ranged from 76.31% to 111.28% of the administeredradioactivity). At 1152 hours, a mean of 87.0% of the totalradioactivity administered (range 73.2 to 107%) was recovered fromfeces. The majority of drug-related material in the feces was recoveredwithin 552 hours of dosing with investigational product. In Part 2 (MassBalance), the mass balance data confirmed that the primary eliminationroute of OCA is through the feces. A mean of 87% of administeredradioactivity was recovered from the feces over the entire collectionperiod, and a minor percentage (<3%) was recovered from the urine.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

1. A compound of Formula I

or a pharmaceutically acceptable salt thereof, wherein R₁ is OH,O-glucuronide, OSO₃H, SO₃H, CO₂R₅, ¹⁴CO₂R₅, C(O)R₆, ¹⁴C(O)R₆; R₂ is H,D, or OH; R₃ is OH or O-glucuronide; R₄ is OH or O-glucuronide; R₅ is Hor substituted or unsubstituted alkyl; R₆ is NH(CH₂)₂SO₃H, NHCH₂CO₂H, orN(CH₃)CH₂CO₂H or glucuronic acid moieties, wherein hydrogens in can bereplaced with deuterium; R₁₁ is Z_(11b), hydroxy, halogen, alkoxy, oroxo when Z_(11a) is not present; R₁₆ is Z_(16a), hydroxy, halogen,alkoxy, or oxo when Z_(16b) is not present; R₂₃ is Z_(23a) or alkyl; andZ_(1a), Z_(1b), Z_(2a), Z_(2b), Z₃, Z_(4a), Z_(4b), Z₅, Z₆, Z₇, Z₈, Z₉,Z_(11a), Z_(11b), Z₁₂, Z₁₄, Z_(15a), Z_(15b), Z_(16a), Z_(16b), Z₁₇,Z₁₈, Z₁₉, Z₂₀, Z₂₁, Z_(22a), Z_(22b), Z_(23a), Z_(23b), Z_(24a),Z_(24b), or Z₂₅ is independently selected from H (hydrogen) or D(deuterium), and at least one of Z_(1a), Z_(1b), Z_(2a), Z_(2b), Z₃,Z_(4a), Z_(4b), Z₅, Z₆, Z₇, Z₈, Z₉, Z_(11a), Z_(11b), Z₁₂, Z₁₄, Z_(15a),Z_(15b), Z_(16a), Z_(16b), Z₁₇, Z₁₈, Z₁₉, Z₂₀, Z₂₁, Z_(22a), Z_(22b),Z_(23a), Z_(23b), Z_(24a), Z_(24b), or Z₂₅ is D; and any carbon atom is¹²C, ¹³C, or ¹⁴C.
 2. The compound of claim 1 wherein R₂ is H.
 3. Thecompound of claim 1 wherein R₂ is OH.
 4. The compound of claim 3 whereinR₂ is alpha-OH.
 5. The compound of claim 1, wherein R₆ is taurine(—NH(CH₂)₂SO₃H), glycine (—NHCH₂CO₂H), or sarcosine (—N(CH₃)CH₂CO₂H). 6.The compound of claim 1, wherein compound of Formula I is a compound ofFormula II


7. The compound of claim 1, wherein compound of Formula I is a compoundof Formula III


8. The compound of claim 1, wherein compound of Formula I is a compoundof Formula IV


9. The compound of claim 1, wherein compound of Formula I is a compoundof Formula V


10. The compound of claim 1, wherein compound of Formula I is a compoundof Formula VI


11. The compound of claim 1, wherein compound of Formula I is a compoundof Formula VII


12. The compound of claim 1, wherein compound of Formula I is a compoundof Formula VIII

wherein R₁ is ¹⁴CO₂R₅ or ¹⁴C(O)R₆.